Variable Lymphocyte Receptors, Related Polypeptides and Nucleic Acids, and Uses Thereof

ABSTRACT

Disclosed are compositions and methods related to variable lymphocyte receptors (VLRs).

The application claims the benefit of U.S. provisional Application60/573,563, filed May 21, 2004, which is incorporated herein byreference in its entirety.

This invention was made with government support under NIH/NIAID GrantAI39816 and HG02526-01 and NSF Grants MCB-0317460 and IBN-0321461. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adaptive immune responses in jawed vertebrates are initiated whenantigens are recognized by specific lymphocyte receptors. Antigenreceptor diversity is generated via recombination of variable, diversityand joining gene segments in the immunoglobulin (Ig) and T cell receptor(TCR) gene loci. This combinatorial rearrangement generates vastrepertoires of antibodies against unprocessed antigens and of TCRs thatrecognize antigen fragments presented within the cusp of majorhistocompatibility complex (MHC) class I and II molecules. Clonallydiverse lymphocytes thus form the cornerstone of vertebrate adaptiveimmunity in the form of Ig bearing B cells and TCR bearing T cells thatdifferentiate from stem cell precursors within primary hematopoietictissues and the thymus. Cardinal elements of this recombinatorial immunesystem are conserved in all jawed vertebrates and the multigene TCR andIg loci are remarkably complex even in the most basal gnathostomerepresentatives, sharks, skates, and rays (Rast et al., 1997; Flajnikand Kasahara, 2001; Flajnik, 2002).

There is also abundant evidence for adaptive immunity in the jawlessvertebrates, lamprey and hagfish, the only surviving descendents fromthe early vertebrate radiation (Forey and Janvier, 1993). Humoral andcell mediated types of immunologic responses have been reported forthese agnathans. For example, lampreys produce specific circulatingagglutinins in response to primary antigenic stimulation, make higheragglutinin levels after booster immunization (Finstad and Good, 1964;Marchalonis and Edelman, 1968; Litman et al., 1970; Pollara et al.,1970; Good et al., 1972; Hagen et al., 1985), reject second set skinallografts at an accelerated rate (Finstad et al., 1964; Perey et al.,1968; Good et al., 1972; Fujii and Hayakawa, 1983) and exhibit delayedtype hypersensitivity reactions (Finstad and Good, 1964; Good et al.,1972). Agnathan adaptive immune responses have been attributed to cellsthat morphologically resemble the lymphocytes found in thelympho-hematopoietic tissues and blood of jawed vertebrates (Finstad andGood, 1964; Finstad et al., 1964; Perey et al., 1968; Cooper, 1971;Piavis and Hiatt, 1971; Good et al., 1972; Kilarski and Plytycz, 1981;Zapata et al., 1981; Fujii, 1982; Fujii and Hayakawa, 1983; Ardavin andZapata, 1987; Mayer et al., 2002a). Like their mammalian counterparts,lamprey lymphocytes are more irradiation sensitive than other blood celltypes (Good et al., 1972), aggregate and proliferate in response toantigenic stimulation (Finstad and Good, 1964; Cooper, 1971; Piavis andHiatt, 1971), and express transcription factors that are involved inmammalian lymphocyte differentiation, such as PU.1/Spi-B and Ikaros(Haire et al., 2000; Shintani et al., 2000; Anderson et al., 2001; Mayeret al., 2002b). Surprisingly, however, Ig, TCR, and MHC genes have notbeen previously identified in jawless vertebrates or in the genomesequence of the invertebrate urochordate Ciona intestinalis (Azumi etal., 2003). The present invention relates to a novel lymphocyte receptorand nucleic acids that encode a novel lymphocyte receptor.

SUMMARY OF THE INVENTION

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates topolypeptides comprising a novel lymphocyte receptor or fragmentsthereof. The invention further relates to nucleic acids that encode thelymphocyte receptors or fragments. Further provided are methods ofmaking and using the polypeptides and nucleic acids. Such uses include abroad range of purification, therapeutic and diagnostic methods.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 shows lamprey leukocytes and VLRs. FIG. 1 a shows a light scatteranalysis of blood leukocytes before and after immunostimulation withantigen/mitogen cocktail. FIG. 1 b shows sorted immunostimulatedleukocytes: small lymphocytes (R1) large lymphocytes (R2) or myeloidcells (R3). Wright-Giemsa stain, 100×. Scale bar=10 μm. FIG. 1 c showsvirtual Northern blots of VLR and GAPDH (control). Amplified cDNA fromtissues or sorted cells from hematopoietic organs and blood ofimmunostimulated and unstimulated larvae are shown. FIG. 1 d shows a VLRstick model: signal peptide, N-terminal LRR, nine LRRs, connectingpeptide, C-terminal LRR, threonine-proline rich stalk, GPI-anchor andhydrophobic tail (Clone 12.26, 417 residues, AY577974). FIG. 1 e showsthe cell surface expression of epitope-tagged VLR and FcγRIIb (control)expressed in mouse thymoma cells, treated with (+PLC) or without (−PLC)bacterial GPI-phospholipase C. FIG. 1 f shows a 3D model of VLRdiversity region viewed in two rotations (clone 12.26).

FIG. 2 shows a survey of VLR diversity in two lamprey larvae. Alignmentof 20 diversity regions PCR amplified from lymphocytes. PCR primers werelocated in regions conserved in all VLR sequences: signal peptide 5′ toLRRNT and near 3′ of LRRCT. Donor animals and clone numbers areindicated. The locations of LRR motifs are also indicated. Black: 100%identity; gray: 60-99%; white: 60%. Sequences 1.3-2.10 correspond hereinto SEQ ID NOs:1-20, respectively.

FIG. 3 shows an assessment of VLR protein diversity in 13 individuallarvae. Genetic distance dendrogram of 112 VLR diversity regions fromcDNA and genomic PCR clones. Larvae numbers and clone numbers (e.g.,6.20=donor 6, clone 20) are indicated in red for immunostimulated (N=27)and green for unstimulated (N=41) donors. Asterisk (*) indicates clonesderived from single cell isolates (N=12), including two VLRs from oneisolate (9.16S, 9.16L); and clones derived from a control 10-cell poolare denoted 10C (N=4). Mature VLR sequences derived from genomic DNA arein blue (N=28; blood #10,12; carcass #11, 13). The mean diversity forthe entire set is 1.36±0.03, ranging 0.28-0.54 within the groups ofsequences from 13 individuals.

FIG. 4 shows VLR genome blots of restriction-enzyme digested DNA thatwere hybridized with VLR N-terminal or C-terminal probes. FIG. 4( a)shows blots of three lampreys (blood DNA #10,12; carcass #13) Onlyanimal 13 showed a polymorphic BamHI pattern. FIG. 4 b shows a genomespread of erythrocytes pooled from 10 lampreys. Pulse-filed blothybridization shows matching patterns for both probes, with anadditional 350 kb Nod N-terminal band corresponding to a 5′ gVLRduplication.

FIG. 5 shows the genomic organization of the VLR locus. FIG. 5 a showsmotifs identified in a 57 kb gVLR contig (AY577941) melded from clonesPAC16 (44 kb) and PAC3 (33 kb) that overlap over 20 kb. Dashed linesrepresent PAC inserts; red bars indicate N-terminal and C-terminalprobes. FIG. 5 b that PAC4 (58 kb, AY577942) aligns with the gVLR contigover 11.7 kb (nt 45,882-57,609). Cassettes of 1-3 LRRs are positioned inforward or reverse orientations: eight in the gVLR contig and 17 inPAC4. FIG. 5 c shows LR-PCR analysis of the gVLR. DNA from blood (#10)or body carcass (#13) amplified with primers gVLR.F1+gVLR.R1 (indicatedin FIG. 5 a and FIG. 5 e). PAC16 amplicon served as control. The ˜20 kbband corresponds to the germline VLR and the ˜8 kb band corresponds tomature VLRs. FIG. 5 d shows lymphocyte specific rearrangement of matureVLRs. LR-PCR from sorted pools of 100 lymphocytes or erythrocytes. The˜14 kb band corresponds to the germline VLR and the ˜1 kb bandcorresponds to mature VLRs that were amplified only from lymphocyte DNA.FIG. 5 e shows an illustration of an 8 kb mature VLR amplicon.

FIG. 6 shows the multiple alignment of 22 VLR proteins predicted fromEST clones (single pass 5′ sequence, some incomplete C-termini). Black:full identity; yellow 80-99%; green: 60-79%; white <60%. The amino acidsequences for LyEST3090-LyEST5266 correspond to SEQ ID NOs:21-42,respectively.

FIG. 7 shows an ORF of a representative VLR (cDNA clone LyEST2913,AY578059). The start methionine is at nt 118-120 and the stop codon atnt 937-939. Nucleotide sequence conserved in exons 2 and 4 of thegermline VLR are colored red; the diverse 5′ LRRCT corresponding to exon3 is colored green. Structural motifs are indicated above the proteinsequence; GPI cleavage site is colored blue. The amino acid sequenceshown corresponds to SEQ ID NO:43, and the nucleic acid sequence showncorresponds to SEQ ID No:156.

FIG. 8 shows the multiple alignment of 112 VLR diversity regions PCRamplified from 13 lampreys. Genomic and RT-PCR clones fromimmunostimulated and unstimulated lampreys. Unstimulated animals: animaldesignated #1-4 (N=41), sorted single lymphocytes from animal designated#8 (N=4) and clones from a pool of 10 cells from animal designated #8.10C(N=4); Immune stimulated animals: from animals designated #5-7 (N=27)and sorted single lymphocytes from animal designated #9 (N=8) includingone isolate with two VLRs (9.16S, 9.16L); Mature VLRs: larval genomicDNA extracted from blood designated #10-13 (N=28) or carcass (#11, 13).Black: 80-100% identity; yellow 60-79%; green: 40-59%; white <40%. Fromthe top of the alignment, the amino acid sequence for 1.1 corresponds toSEQ ID NO:13, amino acid sequences 7.27-4.7 correspond to SEQ IDNOs:45-52, amino acid sequence 1.5 corresponds to SEQ ID NO:12, aminoacid sequence 4.14 corresponds to SEQ ID NO:54, amino acid sequence 1.7corresponds to SEQ ID NO:8, amino acid sequence 3.15 corresponds to SEQID NO:56, amino acid sequence 2.1 corresponds to SEQ ID NO:5, amino acidsequence 2.2 corresponds to 10, amino acid sequence 2.7 corresponds toSEQ ID NO:11, amino acid sequences 4.8-6.22 correspond to SEQ IDNOs:60-65, amino acid sequences 2.4 corresponds to SEQ ID NO:3, aminoacid sequence 1.8 corresponds to SEQ ID NO:2, amino acid sequences7.3-6.21 correspond to SEQ ID NOs:68-72, amino acid sequence 1.2corresponds to SEQ ID NO:5, amino acid sequence 2.14 corresponds to SEQID NO:6, amino acid sequence 3.7 corresponds to SEQ ID NO:75, amino acidsequence 1.6 corresponds to SEQ ID NO:7, amino acid sequence 5.3corresponds to SEQ ID NO:77, amino acid sequence 10.1 corresponds to SEQID NO:78, amino acid sequence 2.14 corresponds to SEQ ID NO:4, aminoacid sequence 1.3 corresponds to SEQ ID NO:1, amino acid sequences6.16-7.26 correspond to SEQ ID NOs:81-119, amino acid sequence 2.15corresponds to SEQ ID NO:14, amino acid sequence 2.8 corresponds to SEQID NO:17, amino acid sequences 5.6-7.33 correspond to SEQ IDNOs:122-125, amino acid sequence 1.10 corresponds to SEQ ID NO:19, aminoacid sequence 2.10 corresponds to SEQ ID NO:20, amino acid sequence 1.4corresponds to SEQ ID NO:15, amino acid sequences 12.19-4.3 correspondto SEQ ID NOs:129-132, amino acid sequence 1.9 corresponds to SEQ IDNO:16, amino acid sequences 5.5-3.3 correspond to SEQ ID NOs:134-144,amino acid sequence 2.13 corresponds to SEQ ID NO:18, and amino acidsequences 3.6-3.9 correspond to SEQ ID NOs:146-155.

FIG. 9 shows the evolutionarily conserved agnathan VLRs. VLR amino acidsequences representing the Inshore hagfish (Eptatretus burgeri), Pacifichagfish (E. stoutii), Sea lamprey (Petromyzon marinus; GenBank accessionAY577946), American brook lamprey (Lampetra appendix) and Northern brooklamprey (Ichthyomyzon fossor). Blue shade: 100% identity; yellow:60-99%; green: 40-59%; red: hydrophobic tail region.

FIG. 10 shows the genetic distance among Pacific hagfish VLR diversityregions (LRRNT to LRRCT). Proteins predicted form PCR amplifiedlymphocyte-like cDNA clones, or blood genomic PCR amplicons from fiveanimals. Scale bars represent 5% amino acid divergence. A. VLR-A(N=139). B. VLR-B (N=70). Green: unstimulated; red: immunostimulated;blue: genomic mature VLR; asterisk—related sequences.

FIG. 11 shows the hagfish VLR gene loci. FIG. 11A shows the Pacifichagfish VLR-A. FIG. 11B shows the Inshore hagfish VLR-A. FIG. 11C showsthe Pacific hagfish VLR-B. FIG. 11D shows the Inshore hagfish VLR-B.Sequence of inserts from four BAC clones, with uncaptured gaps marked.Location of VLR germline genes and flanking cassettes, in reverse orforward orientation, is indicated in kilobases (graphics are out ofscale). GenScan gene predictions indicated in blue: an unrelated LRRgene upstream from the Pacific hagfish germline VLR-A gene and twoflanking transposase ORFs in the Inshore hagfish VLR-A and Pacifichagfish VLR-B loci.

FIG. 12 shows the Agnathan VLR genes, transcripts and phylogeny. FIG.12A shows a schematic presentation of germline and mature VLR genes ofPacific hagfish and Sea lamprey. Colored bars indicate coding regions;size in nucleotides; positions of PCR primers (Table 5) used to amplifyhagfish VLR are indicated by arrows ad labeled F (forward) R (reverse).FIG. 12B shows Pacific hagfish VLRs PCR amplified from lymphocyte-liketranscripts (RT-PCR) or blood genomic DNA. Agarose gel image; molecularweight marker indicated on the left (kilobases); position of germlineand mature VLR amplicons indicated on the right. FIG. 12C shows thephylogenetic analysis of agnathan VLRs. Neighbor Joining tree of hagfishand lamprey VLR proteins (same sequences as in FIG. 9); bootstrap valuesare indicated. Scale bar represents 10% amino acid divergence. FIG. 12Dshows a model for the evolution of agnathan VLR.

FIG. 13 shows the Genetic distance among Inshore hagfish VLR diversityregions (LRRNT to LRRCT). Proteins were predicted from leukocyte cDNAclones, or mature VLR amplicons from genomic DNA of three animals. Scalebars represent 5% amino acid divergence. A. VLR-A (N=66). B. VLR-B(N=18). Red: hagfish #7; green: #8; blue: genomic mature VLR fromhagfish #9.

DETAILED DESCRIPTION

A lymphocentric search was initiated for primordial elements of thevertebrate immune system in the sea lamprey, Petromyzon marinus, amodern representative of the oldest vertebrates. An earlier analysis oftranscripts expressed by lymphocyte-like cells from lampreyhematopoietic tissues identified several homologs of immune systemmolecules (Mayer et al., 2002a; Uinuk-Ool et al., 2002; Uinuk-Ool etal., 2003), but none of the cardinal Ig superfamily receptor elementsemployed by jawed vertebrates for specific adaptive immunity wereidentified. Reasoning that activated lymphoblasts present in the bloodstream were more likely to express the genes involved in adaptiveresponses, the present study began with a survey of the transcriptome ofblood lymphocytes from immunostimulated lamprey larvae. This searchrevealed a novel type of highly variable lymphocyte receptors which aredescribed here.

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific synthetic methods, specificrecombinant biotechnology methods unless otherwise specified, or toparticular reagents unless otherwise specified, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

As used herein, “polypeptide,” “protein,” and “peptide” are usedinterchangeably to refer to amino acid sequences.

The invention relates to a variable lymphocyte receptor (VLR), which isa polypeptide capable of somatic rearrangement, which comprises 1-12leucine rich repeats and which can function in adaptive immunity.

The invention provides an isolated polypeptide comprising an N-terminalleucine rich repeat (LRRNT), one or more leucine rich repeats (LRRs)(referred to herein as the internal LRRs), a C-terminal leucine richrepeat (LRRCT), and a connecting peptide, wherein the connecting peptidecomprises an alpha helix. The length of the polypeptide can comprise asfew as about 130 amino acids or as many as about 225 amino acids.Examples of the general structure and specific sequences of thepolypeptides and encoding nucleic acids are shown in Figures.Furthermore numerous examples of various regions (including the signalpeptide, LRRNT, LRR, LRRCT, connecting peptide, stalk and hydrophobictails) can be found in Figures.

Optionally the connecting peptide is located on the N-terminal side ofthe LRRCT, and more specifically located between the internal LRR andthe LRRCT. The connecting peptide can be linked to an internal LRR andthe LRRCT. Thus disclosed herein are polypeptides comprising a LRRNT,one or more internal LRRs, a connecting peptide, and a LRRCT, in thatorder. Also disclosed are polypeptides, wherein the internal LRR regionbetween the LRRNT and the LRRCT comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9leucine rich repeats, with LRR 1 located adjacent to or close to theLRRNT. As used herein LRRs 1, 2, 3, 4, 5, 6, 7, 8, or 9 are consideredto run from the LRRNT to the LLRCT consecutively. Thus disclosed hereinare polypeptides comprising a LRRNT, 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, or 1-9 LRRs, a connecting peptide, and a LRRCT, in that order.

Leucine rich repeats (LRRs) are short sequence motifs typically involvedin protein to protein interactions, wherein the LRRs comprise multipleleucine residues. LRRs contain leucine or other aliphatic residues, forexample, at positions 2, 5, 7, 12, 16, 21, and 24. However, it isunderstood and herein contemplated that the leucine or other aliphaticresidues can occur at other positions in addition to or in the place ofresidues at positions 2, 5, 7, 12, 16, 21, and 24. For example, aleucine can occur at position 3 rather than position 2. It is alsounderstood that structurally, the motifs form β-sheet structures. Thus,for example, a disclosed polypeptide comprising a LRRNT, 5 LRR, a LRRCT,and a connecting peptide would comprise 7 β-sheet structures and thealpha helix of the connecting peptide.

It is understood that the length and sequence of each LRR can vary fromthe other LRRs in the polypeptide as well as from the LRRNT and LRRCT.For example, one embodiment of the present invention are polypeptidescomprising a LRRNT, 1-9 LRR, a connecting peptides, and a LRRCT, whereinthe first internal LRR is LRR1, and wherein LRR1 comprises less thanabout 20 amino acids. Also disclosed are polypeptides, wherein LRR1comprises about 18 amino acids. Optionally, the polypeptide furthercomprises LRR2-9, wherein LRR2-9 are less than about 25 amino acidseach. Also disclosed are polypeptides, wherein LRR2-9 comprise about 24amino acids each. LRR 1-9 can be the same or different from each otherin a given polypeptide both in length and in specific amino acidsequence.

The terminal LRRs, designated LRRNT and LRRCT, are typically longer thaneach internal LRR. The LRRNT and LRRCT comprise invariant regions(regions that have little variation relative to the rest of thepolypeptide as compared to similar variable lymphocyte receptors). Thevariable regions provide the receptors with specificity, but theinvariant regions and general structural similarities across receptorshelp maintain the protective immunity functions. The polypeptide cancomprise an LRRNT, wherein the LRRNT comprises less than about 40 aminoacids. Thus the LRRNT optionally comprises the amino acid sequenceCPSQCSC (SEQ ID NO: 157), CPSRCSC (SEQ ID NO: 307), CPAQCSC (SEQ ID NO:308), CPSQCLC (SEQ ID NO: 309), CPSQCPC (SEQ ID NO: 310), NGATCKK (SEQID NO: 311), or NEALCKK (SEQ ID NO: 312) in the presence or absence ofone or more conservative amino acid substitutions.

Also disclosed are polypeptides comprising a LRRCT, wherein the LRRCT isless than about 60 amino acids, and optionally 40-60 amino acids inlength. In particular, specifically disclosed are polypeptides, whereinthe LRRCT comprises the amino acid sequence TNTPVRAVTEASTSPSKCP (SEQ IDNO:158), SGKPVRSIICP (SEQ ID NO: 313), SSKAVLDVTEEEAAEDCV (SEQ ID NO:314), or QSKAVLEITEKDAASDCV (SEQ ID NO: 315) in the presence or absenceof conservative amino acid substitutions.

As with all peptides, polypeptides, and proteins, it is understood thatsubstitutions in the amino acid sequence of the LRRCT and LRRNT canoccur that do not alter the nature or function of the peptides,polypeptides, or proteins. Such substitutions include conservative aminoacid substitutions and are discussed in greater detail below.

The disclosed compositions can also comprise a connecting peptide.Typically such peptides are short peptides less than 15 amino acids inlength and comprise an alpha helix. Thus, for example, specificallydisclosed are connecting peptides of 10, 11, 12, 13, 14, and 15 aminoacids in length comprising an alpha helix. It is understood that theconnecting peptide serves to link structural components of thepolypeptide. It is further understood that the connecting peptide of thepolypeptide can be linked to the LRRCT.

The polypeptides of the invention can comprise soluble or membrane boundforms. Many mechanisms exist that allow a polypeptide to be soluble ormembrane bound. For example, a polypeptide missing a transmembranedomain can be secreted directly by a cell. Alternatively, a polypeptidecan comprise a glycosyl-phosphatidyl-inositol (GPI) anchor whichmaintains the polypeptide on a membrane surface. Therefore, disclosedherein are polypeptides comprising a GPI anchor. Other mechanisms formaintaining a polypeptide bound to a surface are known in the art. Forexample, the polypeptide may be bound to a hydrophobic layer throughsingle or multi-pass transmembrane regions that form covalentinteractions with the lipid bilayer of the membrane. Alternatively, thepolypeptide may be bound to the surface through noncovalent interactionswith surface proteins.

The polypeptides of the invention can be surface bound polypeptides.Trafficking to the cell surface can be conducted by means of a signalpeptide which provides a indicator to the intracellular transportmachinery to deliver the polypeptide to the surface of a cell. Thus itis a further embodiment of the invention that the polypeptides of theinvention comprise a signal peptide of the N-terminal of thepolypeptide.

It is understood and herein contemplated that the polypeptides cancomprise a hydrophobic tail.

The polypeptide can comprise a stalk region. The stalk region comprisesa threonin-proline rich region and is optionally present in the membranebound form of the polypeptide, along with the GPI anchor and thehydrophobic tail.

Examples of polypeptides of the invention include those comprising aminoacid sequences of SEQ ID NOs: 1-43, 45-52, 54, 56, 60-65, 68-72, 75,77-78, 81-119, 122-125, 129-132, 134-144, and 146-155. Sequences includeGenBank Accession Numbers AY577941-AY578059 and CK988414-CK988652. Thosesequences comprising the amino acid sequences of SEQ ID NOs:1-20represent examples of full length VLRs. The sequence comprising theamino acid sequence of SEQ ID NO:43 is an example of a full length VLRwith the signal peptide. Additional full length VLRs and fragmentsthereof comprising the amino acid sequences can be found in the figures.Based on the structure taught herein for the polypeptides of theinvention, it will be understood that these sequences are examples of agenus of polypeptides. It is understood that the invention includes fulllength VLRs and fragments thereof.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular polypeptide is disclosed and discussed and anumber of modifications that can be made to a number of polypeptides arediscussed, specifically contemplated is each and every combination andpermutation of polypeptides and the modifications that are possibleunless specifically indicated to the contrary. Thus, if a class ofmolecules A, B, and C are disclosed as well as a class of molecules D,E, and F and an example of a combination molecule, A-D is disclosed,then even if each is not individually recited each is individually andcollectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F,C-D, C-E, and C-F are considered disclosed. Likewise, any subset orcombination of these is also disclosed. Thus, for example, the sub-groupof A-E, B-F, and C-E would be considered disclosed. This concept appliesto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods.

The polypeptides of the invention have a desired function. Thepolypeptides as described herein selectively bind an antigen or anagent, much as an antibody selectively binds an antigen or agent. Thepolypeptides optionally are variable lymphocyte receptors (naturallyoccurring or non-naturally occurring) or fragments or variants thereof.The term “variable lymphocyte receptors” is used herein in a broad senseand, like the term “antibody” includes various versions having variousspecificities. The polypeptides are tested for their desired activityusing the in vitro assays described herein, or by analogous methods,after which their therapeutic, diagnostic or other purificationactivities are tested according to known testing methods.

The polypeptide of the invention can bind an extracellular agent (e.g.,a pathogen) or antigen. Agents or antigens can include but are notlimited to peptides, polypeptides, lipids, glycolipids, and proteins.Agents or antigens can originate from a variety of sources including butnot limited to pathogenic organisms. The binding to an agent or antigenis understood to be selective. By “selectively binding” or “specificallybinding” is meant that is binds one agent or antigen to the partial orcomplete exclusion or other antigens or agents. By “binding” is meant adetectable binding at least about 1.5 times the background of the assaymethod. For selective or specific binding such a detectable binding canbe detected for a given antigen or agent but not a conrol antigen oragent. Thus, disclosed are polypeptides that selectively bind, forexample, a viral, bacterial, fungal, or protozoan antigen or agent.

Thus specifically disclosed are polypeptides, wherein the polypeptidebinds an agent, wherein the agent is a pathogenic agent. Also disclosedare polypeptides of the invention that selectively binds a pathogenicagent, wherein the pathogen is a virus. Many viruses are known to exist.Thus, the virus can be selected from the group of viruses consisting ofHerpes simplex virus type-1, Herpes simplex virus type-2,Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Humanherpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Variola virus,Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus,Coronavirus, Influenza virus A, Influenza virus B, Measles virus,Polyomavirus, Human Papilomavirus, Respiratory syncytial virus,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus,Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus,Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus,Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valleyfever virus, West Nile virus, Rift Valley fever virus, Rotavirus A,Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency cirus,Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, SimianImmunodeficiency virus, Human Immunodeficiency virus type-1, and HumanImmunodeficiency virus type-2.

Also disclosed are polypeptides of the invention, wherein the pathogenis a bacterium. Many bacteria are known to exist. Specificallycontemplated and herein disclosed are polypeptides that selectively binda pathogen, wherein the pathogen is a bacterium selected from the listof bacteria consisting of M. tuberculosis, M. bovis, M. bovis strainBCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M.kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis,Nocardia asteroides, other Nocardia species, Legionella pneumophila,other Legionella species, Salmonella typhi, other Salmonella species,Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurellamultocida, other Pasteurella species, Actinobacillus pleuropneumoniae,Listeria monocytogenes, Listeria ivanovii, Brucella abortus, otherBrucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydiatrachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsialspecies, Ehrlichia species, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillusanthracis, Escherichia coli, Vibrio cholerae, Campylobacter species,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi,other Hemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species.

Also disclosed are polypeptides of the invention that selectively bind apathogen, wherein the pathogen is a protozoan or other parasite. Manyparasitic infections are known to exist. Specifically contemplated andherein disclosed are polypeptides that selectively bind a pathogen,wherein the pathogen is a parasitic infection selected from the groupconsisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodiumvivax, Plasmodium malariae, other Plasmodium species., Trypanosomabrucei, Trypanosoma cruzi, Leishmania major, other Leishmania species.,Schistosoma mansoni, other Schistosoma species., and Entamoebahistolytica.

Also disclosed are polypeptides of the invention that selectively bind apathogen, wherein the pathogen is a fungus. Many fungi are known toexist. Specifically contemplated and herein disclosed are polypeptides,wherein the pathogen is a fungi selected from the group fungi consistingof Candida albicans, Cryptococcus neoformans, Histoplama capsulatum,Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodesbrasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicilliummarneffi, and Alternaria alternata.

The polypeptide of can also selectively bind to toxins. Herein “toxins”refer to any chemical or biological agent that effectively destroys anycell that it (the toxin) contacts. Notable examples of toxins includericin, pertussis toxin, sarin, bacterial endotoxin, toxic shock syndrometoxin 1, cholera toxin, and snake venom toxins. Thus, specificallydiscloses are polypeptides that bind to a toxin.

The polypeptides described herein can be modified and varied so long asthe desired function is maintained. It is understood that one way todefine any known variants and derivatives or those that might arise, ofthe disclosed genes and proteins herein is through defining the variantsand derivatives in terms of homology to specific known sequences. Forexample SED ID NO: 1 sets forth a particular amino acid sequence of thepolypeptide encoded by any number of nucleic acids of the invention.Specifically disclosed are variants of these and other genes andproteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 percent homology to the stated sequence. Those ofskill in the art readily understand how to determine the homology of twoproteins or nucleic acids, such as genes. For example, the homology canbe calculated after aligning the two sequences so that the homology isat its highest level.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

Protein variants and derivatives are well understood to those of skillin the art and in can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Insertions ordinarily will be smaller insertions than those ofamino or carboxyl terminal fusions, for example, on the order of one tofour residues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations alanine Ala Aallosoleucine AIle arginine Arg R asparagine Asn N aspartic acid Asp Dcysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly Ghistidine His H isolelucine Ile I leucine Leu L lysine Lys Kphenylalanine Phe F proline Pro P pyroglutamic acidp pGlu serine Ser Sthreonine Thr T tyrosine Tyr Y tryptophan Trp W valine Val V

TABLE 2 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Ala; Ser Arg; Lys; Gln Asn;Gln; His Asp; Glu Cys; Ser Gln; Asn, Lys Glu; Asp Gly; Pro His; Asn; GlnIle; Leu; Val Leu; Ile; Val Lys; Arg; Gln; Met; Leu; Ile Phe; Met; Leu;Tyr Ser; Thr Thr; Ser Trp; Tyr Tyr; Trp; Phe Val; Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

As used herein, the term “variable lymphocyte receptor” or “variablelymphocyte receptors” can also refer to polypeptides that have beenmodified to have reduced immunogenicity when administered to a subject.For example, human amino acid sequences may be inserted within or addedto the polypeptide to make a version less immunogenic to a humansubject, much like antibodies are humanized. Many non-human variablelymphocyte receptors (e.g., those derived from lampreys, mice, rats, orrabbits) can be naturally antigenic in humans, and thus can give rise toundesirable immune responses when administered to humans. Therefore, theuse of modified polypeptides in the methods of the invention can serveto lessen the chance that a polypeptide administered to a human willevoke an undesirable immune response.

Modification techniques can involve the use of recombinant DNAtechnology to manipulate the DNA sequence encoding one or morepolypeptide regions of the variable lymphocyte receptor molecule.Accordingly, the humanized form of the variable lymphocyte receptor (ora fragment thereof) is a chimeric variable lymphocyte receptor,preferably the antigen (agent)-binding portion of the variablelymphocyte receptor) which contains a portion of an antigen (agent)binding site from a non-human (donor) variable lymphocyte receptorintegrated into human (recipient) amino acid sequence.

It is understood that the nucleic acids that can encode those proteinsequences, variants and fragments thereof are also disclosed. This wouldinclude all degenerate sequences related to a specific protein sequence,i.e. all nucleic acids having a sequence that encodes one particularprotein sequence as well as all nucleic acids, including degeneratenucleic acids, encoding the disclosed variants and derivatives of theprotein sequences. Thus, while each particular nucleic acid sequence maynot be written out herein, it is understood that each and every sequenceis in fact disclosed and described herein through the disclosed proteinsequence.

Humanized variable lymphocyte receptors can also contain amino acidsequences which are found neither in the recipient variable lymphocytereceptor nor in the imported human sequences.

The polypeptides of the invention can also used to make fusion proteins.The polypeptides can serve a targeting function in the fusion protein.Thus the polypeptide of the invention can be conjugated to or otherwiselinked by recombinant engineering to a second moiety. The second moietycan comprise a toxin, for example, if cell killing is desired. Thus, forexample, the polypeptide that selectively binds a protozoan can targetthe protozoan and the toxin moiety of the fusion protein can kill thecell. Similarly, the polypeptide of the invention can perform a deliveryfunction. Thus the second moiety can be a therapeutic agent.

The polypeptide of the invention can be linked to a detectable tag. A“detectable tag” is any tag that can be visualized with imaging ordetection methods, in vivo or in vitro. The detectable tag can be aradio-opaque substance, radiolabel, a chemoluminescent label, afluorescent label, or a magnetic label. The detectable tag can beselected from the group consisting of gamma-emitters, beta-emitters, andalpha-emitters, gamma-emitters, positron-emitters, X-ray-emitters andfluorescence-emitters. Suitable fluorescent compounds includefluorescein sodium, fluorescein isothiocyanate, phycoerythrin, and TexasRed sulfonyl chloride, Allophycocyanin (APC), Cy5-PE, CY7-APC, andCascade yellow.

Suitable radioisotopes for labeling include Iodine-131, Iodine-123,Iodine-125, Iodine-126, Iodine-133, Bromine-77, Indium-111, Indium-113m,Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium-103,Ruthenium-105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105,Rhenium-101, Tellurium-121m, Tellurium-122m, Tellurium-125m,Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18.

Optionally the detectable tag can be visualized using histochemicaltechniques, ELISA-like assays, confocal microscopy, fluorescentdetection, cell sorting methods, nuclear magnetic resonance,radioimmunoscintigraphy, X-radiography, positron emission tomography,computerized axial tomography, magnetic resonance imaging, andultrasonography.

Alternatively, the polypeptide can be biotintylated and a subsequentdetectable label like a fluorescently labeled strepavidin can be used toindirectly detect the polypeptide. Biotin is detected by any one ofseveral techniques known in the art. For example, the biotin isdetectable by binding with a fluorescence-labeled avidin and the avidinis labeled with a phycoerythrin or a catenated fluorescent label toincrease the signal associate with each binding event.

Optionally the polypeptide is bound to a solid support such as a slide,a culture dish, a multiwell plate, column, chip, array or stable beads.An “array” includes one or more multiwell arraying means such asmicroplates or slides.

Optionally the polypeptide is bound to a mobile solid support, e.g.,beads, which can be sorted using cell sorting technology. “Mobile solidsupport” refers to a set of distinguishably labeled microspheres orbeads. Preferably, the microspheres are polystyrene-divinylbenzenebeads. Sets of microspheres marked with specific fluorescent dyes andhaving specific fluorescent profiles can be obtained commercially, forexample, from Luminex Corporation (Austin, Tex.).

The invention also provides a plurality of polypeptides of theinvention. Optionally the LRRs of the polypeptides are highly variableacross polypeptides. Thus, the plurality can include polypeptides withdifferent binding specificities, based on the variability of theinternal LRRs.

Also provided are kits that include a container with polypeptides of theinvention or a stable or mobile solid support with polypeptides of theinvention. Optionally the polypeptides are bound to the solid support orthe kit. Optionally the kit contains the polypeptides the sold support,and a linking means for binding the polypeptide to the solid support.

The invention provides isolated nucleic acids that encode thepolypeptides of the invention. One example of such a nucleic acidcomprises the nucleotide sequence of SEQ ID NO:156, the ORF of arepresentative VLR. Other examples of nucleic acids that encode VLRs orfragments thereof include SEQ ID NO:44, SEQ ID NO:53-55, SEQ IDNO:57-59, SEQ ID NO:66-67, SEQ ID NO:73-74, SEQ ID NO:76, SEQ IDNOs:79-80, and SEQ ID NOs:172-302. There are a variety of sequencesrelated to the VLR gene having Genbank Accession NumbersAY57791-AY578059, AY964719-AY964931, AY965520-AY965612,AY965658-AY965681, and CK988414-CK988652. These sequences are hereinincorporated by reference in their entireties as well as for individualsubsequences (regions or fragments) contained therein.

Such nucleic acid sequences are provided by way of example of the genusof nucleic acids and are not intended to be limiting. Also provided areexpression vectors comprising these nucleic acids, wherein the nucleicacids are operably linked to an expression control sequence. Furtherprovided are cultured cells comprising the expression vectors. Suchexpression vectors and cultured cells can be used to make thepolypeptides of the invention.

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for exampleVLR or fragments or variants thereof. The disclosed nucleic acids aremade up of nucleotides, nucleotide analogs, or nucleotide substitutes.

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to the base moiety would include natural and syntheticmodifications of A, C, G, and T/U as well as different purine orpyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftentime base modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxy ribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)CH₃, —O(CH₂)_(n)—ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but may also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science, 1991, 254, 1497-1500).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach thepreparation of such conjugates and include, but are not limited to U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

Disclosed are compositions including primers and probes, which arecapable of interacting with the VLR gene, or comparable genes. Incertain embodiments the primers are used to support DNA amplificationreactions. Typically the primers will be capable of being extended in asequence specific manner. Extension of a primer in a sequence specificmanner includes any methods wherein the sequence and/or composition ofthe nucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner.

The size of the primers or probes for interaction with the VLR gene incertain embodiments can be any size that supports the desired enzymaticmanipulation of the primer, such as DNA amplification or the simplehybridization of the probe or primer. A typical VLR primer or probewould be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750,3000, 3500, or 4000 nucleotides long.

The polypeptides and nucleic acids of the invention can be used in avariety of techniques. For example, the polypeptides can be used todetect a selected agent, to block the activity of a selected agent, topurify an agent, as an imaging tool, and as a therapeutic agent.

Provided herein are methods of detecting an agent in a sample,comprising the steps of contacting the sample with the polypeptide,under conditions in which the polypeptide can bind to the agent in thesample, and detecting the polypeptide bound to the agent in the sample.The bound polypeptide indicates the agent in the sample. Detectionmethods are well known in the art. For example, the polypeptide can belabeled with a detectable tag as described above. The diction method canbe used to note the presence or absence of an agent in the sample. Thedetection method, however, can be further combined with quantificationmethods. In vitro assay methods include colorometric assays such asELISA that allow the quantification of the agent based on a comparisonto a control sample or samples of known agent quantity which can be usedto establish an amount relative to a standard. The methods can alsoinclude radiometric assays that allow for quantification based onemitted radiation and fluorescent assays or any means of visualizationand quantification described above.

The sample can be any sample to be tested including any biologic sample.Samples can include fluid samples (like water, blood, urine, etc.),tissue samples, culture samples, cellular samples, etc.

The polypeptides of the invention may also be used to block the activityof any agent to which it binds, comparable to a blocking antibody. Thusalso disclosed are methods of blocking the activity of an agent,comprising contacting the agent with the polypeptide of the inventionunder conditions for the polypeptide to bind the agent. The binding ofthe polypeptide to the agent blocks the activity of the agent. Thecontacting step can be in vivo or in vitro. Thus, for example, to reducecontamination of a sample, a polypeptide that binds a toxin can be addedto the sample and block the toxin activity.

The polypeptides of the invention may also be used to promote theactivity of an agent to which it binds, comparable to an agonisticantibody. Thus also disclosed are methods of promoting the activity ofan agent, comprising contacting the agent with the polypeptide of theinvention under conditions for the polypeptide to bind the agent. Thebinding of the polypeptide to the agent promotes the activity of theagent.

The polypeptides disclosed herein can be used to determine the functionof a gene with unknown function. Thus, disclosed herein are methods ofusing the disclosed polypeptides in protein knock-down assays. Forexample, the disclosed polypeptides can be expressed in the cytoplasm ofa cell which comprises a gene of unknown function. When the RNAtranscript is being translated in the cytoplasm of the cell, thedisclosed polypeptides can bind the protein product of the genequestion. By monitoring the effect the loss of protein expression has onthe cell, the proteins function can be determined. Thus, specificallydisclosed are polypeptides specific for a gene product of unknownfunction. Also are methods of determining the function of a genecomprising introducing a polypeptide specific for the protein product ofthe gene into the cytoplasm of a cell expressing the gene and monitoringthe effect due to the loss of protein product of the gene with unknownfunction.

The polypeptides of the invention can also be used in imaging methods.For example, the invention provides an imaging method comprisingadministering to a subject an effective amount of the polypeptide anddetecting the localization of the bound polypeptide in the subject.Examples of imaging methods are described above.

The invention also provides methods of purification. Disclosed hereinare methods of purifying an agent from a sample comprising contactingthe sample with a polypeptide under conditions for the polypeptide tobind the agent and form a polypeptide/agent complex; and isolating theagent from the polypeptide/agent complex. For example, the polypeptidecan be bound to a column and the sample can be passed through the columnunder conditions that allow the agent in the sample to bind to the boundpolypeptide. The agent can subsequently be eluted from the column in adesired eluant. The purification methods would be useful as researchmethods and as commercial methods. For example, such a method would beuseful in removing contaminants from pharmacological compounds.

The polypeptides can also be used in therapeutic methods. For example,provided herein is a method of reducing or preventing a pathogeniceffect in a subject comprising administering to the subject an effectiveamount of a polypeptide that binds a pathogen. Also provided is a methodof blocking or promoting the activity of an agent so as to reducedeleterious effects or promote positive effects.

Provided herein are composition comprising the polypeptides or nucleicacids of the invention and a pharmaceutically acceptable carrier. Thecompositions of the invention can also be administered in vivo. Thecompositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. The latter may beeffective when a large number of animals is to be treatedsimultaneously. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the polypeptide of the invention,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art. Suitable carriers andtheir formulations are described in Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers containing the variable lymphocyte receptor,which matrices are in the form of shaped articles, e.g., films,liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of variable lymphocyte receptor being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously, for example. Other compounds will beadministered according to standard procedures used by those skilled inthe art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptomsdisorder are effected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any contraindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

The variable lymphocyte receptors and variable lymphocyte receptorfragments and variants of the invention can also be administered topatients or subjects as a nucleic acid preparation (e.g., DNA or RNA)that encodes the variable lymphocyte receptor or variable lymphocytereceptor fragment or variant, such that the patient's or subject's owncells take up the nucleic acid and produce and secrete the encodedvariable lymphocyte receptor or variable lymphocyte receptor fragment.

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991). Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

Transfer vectors can be any nucleotide construction used to delivernucleic acids into cells (e.g., a plasmid), or as part of a generalstrategy to deliver genes, e.g., as part of recombinant retrovirus oradenovirus (Ram et al. Cancer Res. 53:83-88, (1993)). As used herein,plasmid or viral vectors are agents that transport the disclosed nucleicacids, such as VLR into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. Viral vectors are, for example, Adenovirus, Adeno-associatedvirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronaltrophic virus, Sindbis and other RNA viruses, including these viruseswith the HIV backbone. Also preferred are any viral families which sharethe properties of these viruses which make them suitable for use asvectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, andretroviruses that express the desirable properties of MMLV as a vector.Retroviral vectors are able to carry a larger genetic payload, i.e., atransgene or marker gene, than other viral vectors, and for this reasonare a commonly used vector. However, they are not as useful innon-proliferating cells. Adenovirus vectors are relatively stable andeasy to work with, have high titers, and can be delivered in aerosolformulation, and can transfect non-dividing cells. Pox viral vectors arelarge and have several sites for inserting genes, they are thermostableand can be stored at room temperature. A preferred embodiment is a viralvector which has been engineered so as to suppress the immune responseof the host organism, elicited by the viral antigens. Preferred vectorsof this type will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B 19 coding regions have been deleted, resultingin a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorproated by reference for material related tothe AAV vector.

The vectors of the present invention thus provide DNA molecules whichare capable of integration into a mammalian chromosome withoutsubstantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson., Curr Opin Mol Ther 5: 633-644, 1999). These large DNAviruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), havethe potential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA>220 kb and to infect cells that canstably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedvectors for example, lipids such as liposomes, such as cationicliposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.Liposomes can further comprise proteins to facilitate targeting aparticular cell, if desired. Administration of a composition comprisinga compound and a cationic liposome can be administered to the bloodafferent to a target organ or inhaled into the respiratory tract totarget cells of the respiratory tract. Regarding liposomes, see, e.g.,Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner etal. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a componentof a microcapsule that can be targeted to specific cell types, such asmacrophages, or where the diffusion of the compound or delivery of thecompound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via VLRs, antibodies, receptors, or receptorligands. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Senter, et al.,Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer,60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988);Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al.,Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody or VLR conjugated liposomes (including lipid mediateddrug targeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTF.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or RNA, or the nucleic acidscan be in a vector for delivering the nucleic acids to the cells,whereby the antibody-encoding DNA fragment is under the transcriptionalregulation of a promoter, as would be well understood by one of ordinaryskill in the art. The vector can be a commercially availablepreparation, such as an adenovirus vector (Quantum Biotechnologies, Inc.(Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cellscan be via a variety of mechanisms. As one example, delivery can be viaa liposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof) of the invention. The exact method ofintroducing the altered nucleic acid into mammalian cells is, of course,not limited to the use of retroviral vectors. Other techniques arewidely available for this procedure including the use of adenoviralvectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994),adeno-associated viral (AAV) vectors (Goodman et al., Blood84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al.,Exper. Hematol. 24:738-747, 1996). Physical transduction techniques canalso be used, such as liposome delivery and receptor-mediated and otherendocytosis mechanisms (see, for example, Schwartzenberger et al., Blood87:472-478, 1996). This invention can be used in conjunction with any ofthese or other commonly used gene transfer methods.

As one example, if the antibody-encoding nucleic acid of the inventionis delivered to the cells of a subject in an adenovirus vector, thedosage for administration of adenovirus to humans can range from about10⁷ to 10⁹ plaque forming units (pfu) per injection but can be as highas 10¹² pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997;Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997). A subject canreceive a single injection, or, if additional injections are necessary,they can be repeated at six month intervals (or other appropriate timeintervals, as determined by the skilled practitioner) for an indefiniteperiod and/or until the efficacy of the treatment has been established.

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein. For additional discussion of suitable formulations and variousroutes of administration of therapeutic compounds, see, e.g., Remington:The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995.

The invention further provides a method of making a polypeptide of theinvention comprising culturing a cell comprising a vector comprising anucleic acid that encodes the polypeptide and purifying the polypeptidefrom the cell or from the medium. Further provided are methods of makinga polypeptide of the invention using protein synthesis techniques.

Also disclosed are methods of screening for one or more variablelymphocyte receptors in a subject comprising identifying in the subjectone or more polypeptides comprising an N-terminal leucine rich repeat(LRRNT), one or more leucine rich repeats (LRRs), a C-terminal leucinerich repeat (LRRCT), and a connecting peptide, wherein the connectingpeptide comprises an alpha helix.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Variable Lymphocyte Receptors in Sea Lamprey Analysis ofTranscripts from Immunostimulated Blood Lymphocytes

In order to survey the transcriptome of activated lymphocytes, lampreylarvae were stimulated by intraperitoneal injections of anantigen/mitogen cocktail, including live E. coli bacteria, sheeperythrocytes, phytohemagglutinin and pokeweed mitogen, two to four timesat weekly intervals. The fraction of large lymphocytes among peripheralblood leukocytes three days after the second booster stimulation was13-fold greater than in unstimulated individuals, and the fraction ofmyeloid cells was also 6-fold greater (FIG. 1 a). Compared to the smallblood lymphocytes, the large lymphocytes were nearly double in size, hadextensive azurophilic cytoplasm and featured prominent nucleoli (FIG. 1b). These cells were sorted and used to construct cDNA librariesenriched in messages of activated lymphocytes by subtraction againstcDNA from lamprey activated myeloid cells or erythrocytes.

The most abundant group of sequences identified among 1,507 clones fromthe subtracted libraries predicted 319 proteins with variable numbers ofdiverse leucine-rich repeat (LRR) motifs, that clustered with a set of52 LRR-containing expressed sequence tags (EST) from a survey ofunstimulated lymphocyte transcripts. After purging the 3′ end sequences,a set of 239 uniquely diverse LRR proteins were identified, 22 of whichencoded most or all of the open reading frames (ORF) of 239-304 aa (FIG.6). These lamprey proteins were provisionally named variable lymphocytereceptors (VLR) because each of these 239 sequences was unique and theirtranscripts were found to be expressed predominantly by lymphocytes(FIG. 1 c). Lymphocytes from hematopoietic tissues showed highest VLRlevels in unstimulated animals, and immune stimulation resulted inenhanced VLR transcription by the large blood lymphocytes. The basiccomposition of these VLRs included a conserved signal peptide,N-terminal LRR (LRRNT), a variable number of diverse LRRs, a connectingpeptide followed by a C-terminal LRR (LRRCT) and a conserved C-terminuscomposed of a threonine- and praline rich stalk, a genericglycosyl-phosphatidyl-inositol (GPI)-anchor site and a hydrophobic tail(FIG. 1 d and FIG. 7). When a retroviral construct encoding an epitopetagged VLR was transfected into a mammalian cell line,immunofluorescence analysis confirmed the cell surface localization ofthe protein, and treatment with bacterial GPI-specific phospholipase Csignificantly reduced the level of cell surface expression (FIG. 1 e)and released VLR protein into the supernatant. The longest VLR sequenceconsisting of 11 LRRs was threaded on the crystal structure coordinatesof related LRR proteins to generate a 3-dimensional structural model(Schwede et al., 2000). The model provides a concave solenoid structurein which nine β-sheets are capped on both ends by the LRRNT and LRRCT(FIG. 1 f), similar to the model predicted for Toll-like receptor (TLR)ectodomains (Bell et al., 2003).

The VLR Repertoire is Highly Diverse in Individual Lampreys

The VLR diversity was surveyed in individual lampreys by RT-PCR. Bloodleukocytes mRNA from three immunostimulated and four unstimulated larvaewas amplified with primers flanking the VLRs diversity region.Sequencing of ˜10 clones per animal yielded 69 unique VLRs and only twoidentical clones from one individual. Variable sequences of 20 VLRs fromtwo animals illustrate the protein diversity (FIG. 2; entire setincluded in FIG. 3 and FIG. 8). The size variation, 134-214 aa, isprimarily due to differences in number of LRR modules. Each sequencecontains an LRRNT, an 18 aa LRR1, 1-9 LRRs almost invariably 24 aa long,a 13 aa connecting peptide and C-terminal LRRCT; the LRRNTs have 30-38aa and the LRRCTs 48-58 aa. While regions of pronounced sequencediversity are evident for each LRR motif, the first seven residues inLRRNT and the last 20 residues in LRRCT are nearly invariant.

To assess VLRs diversity at the level of individual lymphocytes RT-PCRwith primers flanking the whole ORF was used. Single cell isolates weresorted from the blood of an immunostimulated and an unstimulated larvae.Analysis of the PCR products obtained from six single cell reactionsfrom the unstimulated animal and seven reactions from theimmunostimulated larva showed that 12 of the 13 lymphocytes expressed asingle VLR (FIG. 3), and five of six VLR clones from a control pool of10 unstimulated cells were unique. One cell isolate yielded two VLRs(9.16S, 9.16L), but the possibility that this isolate contained twolymphocytes cannot be excluded. Three of the VLRs had in-frame stopcodons predicting truncated proteins. Interestingly, combinations ofidentical VLRs were identified among five lymphocytes from theimmunostimulated larva (9.1=9.16S; 9.2=9.16L; 9.7=9.9). The analysis ofblood samples from three additional immunostimulated larvae (#5-7)revealed only unique VLRs (N=27). These findings are indicative ofmonoallelic expression of the diverse VLRs, and provide preliminaryevidence for clonal expansion of VLR-bearing lymphocytes.

Complexity of the VLR Locus

Genome blot hybridization with a conserved C-terminal probe revealed asingle band (FIG. 4 a). The N-terminal probe, consisting of theconserved 5′ UTR and signal peptide, reacted with 2-3 bands dependingupon the restriction enzyme employed, except for an individual whoseblot showed 2 additional BamHI bands. In addition, a genomic pulse-fieldCHEF blot revealed a single hybridization band with the C-terminal probein all six digests, whereas the N-terminal probe produced a matchingpattern with one additional 350 kb NotI band (FIG. 4 b). These findingsindicate a single VLR locus, with the N-terminus and C-terminus of thegermline VLR gene (gVLR) contained within 100-150 kb of the genome (FIG.4 b; PacI digest). To further characterize the locus, these probes wereused to screen a large insert sea lamprey P1 bacterial artificialchromosome (PAC) library constructed from erythrocyte DNA of one adult.In an analysis of five PACs that hybridized with both probes, a single14 kb VLR gene (gVLR) amplicon was identified by long range PCR (LR-PCR)using the ORF-flanking primers. Restriction-enzyme analysis of the PCRproducts revealed identical EcoRI bands and two allelic BamHI patterns.PAC clones representing the two gVLR alleles were sequenced, PAC3 andPAC16 with 33 and 44 kb inserts respectively. Their sequences overlappeda 20 kb region containing the gVLR; PAC16 extended 25 kb upstream fromthe gVLR and PAC3 extended 18 kb downstream. The overlap region betweenPACs 3 and 16 was nearly identical, except for short deletions in thegVLR of PAC16 (24, 43 and 78 bp). These sequences were therefore meldedinto a gVLR contig preserving the slightly longer sequence of PAC3 (FIG.5 a).

The gVLR in the PAC3/16 contig consist of 4 exons. The first containspart of the 5′ UTR; exon 2 contains the rest of the 5′ UTR, a signalpeptide and the 5′ half of LRRNT; exon 3 encodes the 5′ half of LRRCT,and exon 4 encodes the 3′ half of LRRCT, the C-terminus and 3′ UTR.Canonical eukaryotic splice sites were identified only in the 5′ UTRintron, while other exon/intron boundaries in the gVLR were determinedby alignment to cDNA sequences. Notably, the gVLR sequence did notcontain a 3′ LRRNT, LRR1 or any of the 24 aa LRRs. Upstream from thisgVLR, six cassettes of variable LRR modules were identified, singlet ordoublet, including LRRNT, LRR1 and LRR positioned either in forward orreverse orientation. These LRR cassettes spanned the first 6 kb of thecontig, while two diverse 5′ LRRCT cassettes were located 7 kbdownstream from the gVLR.

Another clone, PAC4, hybridized only with the N-terminal probe but itwas found to encode multiple LRRs that were identified by PCR with LRRNTand LRR1 consensus primers. The entire insert was 58 kb long (FIG. 5 b),and the sequence overlapped 11.7 kb of the gVLR contig with minor gaps(four gaps of 210-738 bp in PAC4 and eight gaps of 25-55 bp in thePAC3/16 contig). The overlap extended into the intervening sequencebetween gVLR exons 2 and 3, but the 553 bp terminal sequence of PAC4 wasunique. Seventeen cassettes of 1-3 diverse LRR modules, 30 in total,were encoded in a 31 kb region in PAC4 located 15 kb upstream from thepartial gVLR. Comparison of the PAC3/16 gVLR contig and PAC4 sequencesrevealed additional 1-5 kb regions with >90% identity, but these weredisrupted by unrelated sequences. PAC4 could represent either aduplication of ˜12 kb, encompassing the 5′ flank and about half of thegVLR, or a highly divergent VLR allele. To distinguish between thesepossibilities the pattern of genomic hybridization was compared with theN-terminal probe (FIG. 4 a) to the map of restriction sites in the gVLRsfrom these PAC inserts. The blot pattern and restriction map werecompatible for all fragments except for a 5.7 kb HindIII fragment fromPAC4 that was different than the 2 kb band in the blot (FIG. 4 a). Inview of such limited variability amongst three blotted genomes and thegenome from the PAC library, PAC4 seems unlikely to represent apolymorphic gVLR allele. Limited VLR allelic variation would beconsistent with other evidence of low allelic diversity even inmicrosatellite loci (Bryan et al., 2003), indicating the sea lampreypopulations in the North American Great Lakes and other landlockedpopulations are highly inbred. The analysis thus indicates the singlelamprey gVLR locus harbors an additional copy of the N-terminal half ofthe gVLR.

Somatic gVLR Rearrangement Generates Diverse Mature VLRs

When larval DNA samples were analyzed by PCR amplification with primersflanking the VLR diversity region, six unique intron-less VLR ORFs wereobtained (FIG. 3, animals #10, 12). In accordance with this intriguingfinding, PCR amplification of larval DNA samples with the ORFflankingprimers produced VLR clones of 1.5-2 kb including the 5′ UTR intron,revealing unique sequence in 13 of 14 clones (#10, 11). Because thesegenomic PCR clones contained uninterrupted VLR ORFs, they wereprovisionally named mature VLRs to distinguish them from the‘incomplete’ germline VLR. Sequence analysis indicated that these matureVLRs should generate 1-1.3 kb polymorphic EcoRI bands hybridizing withthe N-terminal probe, but these bands were observed only in a lymphocyteDNA blot (FIG. 4 c to be included). These observations indicate thatlamprey DNA samples extracted from pelleted blood erythrocytes or wholelarval bodies contain mature VLRs, but only copies of the germline VLRare sufficiently abundant to be detected in DNA blots from thesesamples.

To address this enigma it was theorized that somatic gene rearrangementin lamprey lymphocytes generated the small mature VLRs, replacingnon-coding DNA from the germline gVLR with diverse LRRs from theupstream and downstream cassettes. To test this hypothesis primers weredesigned for PCR amplification across the germline gVLR, including ˜3 kbof upstream and ˜3 kb of downstream flanks (FIG. 5 a). LR-PCRamplification from larval DNA samples yielded a minor band of ˜20 kb,similar to the gVLR amplicon from PAC16 plasmid, plus an additionalprominent band of ˜8 kb (FIG. 5 c). Sequence analysis of the 8 kbamplicons from two larval samples revealed 9 of 10 clones encodingunique mature VLRs (FIG. 3), the flanks of which were identical to thoseof the gVLR (FIG. 5 d). Altogether 28 unique mature VLRs were identifiedamong the PCR products from four larval DNA samples. Lymphocyte DNA wasmost likely the template for these mature VLRs, as a small fraction ofthe pelleted erythrocytes or whole larval bodies used to extract theseDNA samples. Apparently, the shorter templates of lymphocyte mature VLRswere preferentially amplified during the LR-PCR. A similar PCR bias wasobserved when amplifying with primers that flanked the gVLR ORF,resulting in two amplicons, the 1.5-2 kb of mature VLRs and the 14 kbgVLRs.

The search for lymphocyte receptors that could trigger adaptive immuneresponses in lampreys thus identifies a system of variable lymphocytereceptors that is entirely different from the Ig and TCR of jawedvertebrates. The VLRs consist of multiple LRR modules and an invariantstalk region that is attached to the lymphocyte plasma membrane via aGPI-anchor. The flanking tips of the N-terminal and C-terminal LRRs areinvariant and the remarkable VLR diversity is contributed by variationin number and sequences of the intervening LRRs. The potential VLRdiversity is vast, with 345 out of 354 unique sequences, and only threepairs of identical VLRs from immunostimulated lymphocytes and threeother nearly identical VLRs. The VLRs thus endow this agnathanrepresentative with a diverse repertoire of lymphocyte receptors.

These highly diverse VLRs serve a role in recognition of pathogens.Proteins featuring diverse LRR modules are cardinal innate immunereceptors of animals and plants due to their propensity to interact withan extraordinary vast array of ligands. Animal TLRs are implicated inrecognition of conserved epitopes on viruses, bacteria, fungi andprotozoa, activating signal transduction cascades that culminate ininflammatory responses (Beutler, 2004). CD14, a GPI-anchored LRR proteinthat is also found in a soluble form, binds bacterial lipopolysaccharideand phospholipids to form a signaling complex with the TLR4 receptor(Landmann, 2000). Yet another mammalian family of cytosolic LRRproteins, the NBS-LRRs, recognize intracellular pathogens (Chamaillardet al., 2003). Plant disease resistance genes are members of largemultigene families including hundreds of NBS-LRR proteins,LRR-receptorlike kinases and LRR-receptor-like proteins, many of whichhave been shown to be involved in specific activation of anti-pathogenresponses (Jones et al., 2004). Antigen-binding VLRs with theirremarkable diversity mediate the adaptive immune responses observed inlampreys. The GPI-anchorage of VLRs to the surface of lymphocytes allowGPI-specific phospholipase release of these receptors (Ikezawa 2002),endowing VLRs with dual functionality both as surface receptors andhumoral agglutinins in an anticipatory immune system.

Sequencing genomic PAC clones a germline gVLR consisting of 4 exons thatencoded only the signal peptide, 5′ LRRNT, 5′ LRRCT, 3′ LRRCT and theC-terminus was identified. The gVLR lacked diversity LRR modules exceptfor a 5′ LRRCT, indicating that without modification it could not encodethe highly diverse VLR messages. However, multiple diverse LRR cassetteswere found upstream and downstream from the gVLR, and these could beavailable for insertion into the gVLR to assemble mature VLR genes. Totest the hypothesis that mature VLRs are generated through somaticreplacement of non-coding DNA in the germline gVLR with upstream anddownstream LRR cassettes, LR-PCR was used to detect the presence of bothgermline and mature VLR genes. The expected product of ˜20 kb from thegVLR was obtained from genomic DNA of two lampreys and in addition, thepredicted 8 kb amplicon from mature VLRs, that was found to encode adiverse set of mature VLRs. Moreover, in a few cases candidate LRRdonors could be identified among the gVLR neighboring cassettes based onidentity to VLR sequences, and the highly conserved sequences in thegVLR 5′ LRRNT and 3′ LRRCT could potentially serve as anchoring regionsfor a gene conversion process. VLRs are generated by a mechanism ofsomatic DNA rearrangement.

Non-meiotic DNA rearrangements are known from other systems. Forexample, rearrangement of genes encoding surface components is astrategy used by several pathogens to evade immune recognition duringchronic infection. Antigenic variation in the pilin of Neisseriagonorrhoeae involves non-reciprocal recombination between the pilE locusand multiple silent pilS copies (Hamrick, 2001), and antigenic variationin Lyme disease Borrelia spirochaetes is generated by gene conversionbetween an array of 15 silent cassettes and the vlsE expression site(Wang et al., 2003). Also the protozoan Trypanosoma brucei alternateexpression of their variant surface coat glycoprotein by repeated DNArearrangements (Donelson, 2003), as well as the malaria parasitePlasmodium falciparum and the intestinal dweller Giardia lamblia thatfrequently switch among multiple surface antigen genes. In theevolutionary arms race between hosts and parasites, vertebrates adopteda similar strategy to combat infectious disease by somatic rearrangementof germline receptors. Diverse lymphocyte antigen receptors areassembled via the cut-and-paste activity of the paired transposase-likeRAG1 and RAG2 in gnathostomes (Schluter et al., 1999) and via an as yetuncharacterized mechanism in agnatha.

Features of the lamprey VLR system bear analogy to the Ig and TCR ofjawed vertebrate lymphocytes, with two notable differences. First,lamprey VLRs consist of LRR modules whereas gnathostome antigenreceptors consist of Ig domains. Lampreys immunity underwent a gradualevolutionary process, replacing the ancestral germline encoded diversityof LRR receptors with a system of variable lymphocyte LRR receptors thatare somatically diversified versions of their germline VLR gene. Incontrast, Ig domains as core components of jawed vertebratesrecombinatorial lymphocyte receptors is an intriguing untraceableevolutionary drift from their predecessors, since no Ig superfamilymember has yet been shown to play a role in any type of immunerecognition of pathogens or allografts in animals other than the jawedvertebrates (Kaufman, 2002). Second, no evidence for the existence ofMHC molecules in the lamprey has been found. In jawed vertebratespolymorphic MHC molecules are essential for efficient presentation ofantigen peptides to T-cells, whereas inbred MHC homozygotes appear tosuffer from impaired disease resistance (Penn et al., 2002; Grimholt etal., 2003). Since lampreys thrive as an inbred population in the GreatLakes, this indicates their VLR system may have evolved to functionindependent of polymorphic components.

Animals

Larvae (8-13 cm long) of the sea lamprey were from tributaries to LakeMichigan (Lamprey Services, Ludington, Mich.), or tributaries to LakeHuron (Hammond Bay Biological Station, Millersburg, Mich.). Larvae forimmunostimulation were sedated (100 mg/l MS222; Sigma) and injectedintraperitoneally with 75 μl 0.67X PBS containing: 107 E. coliBL21(DE3), 107 sheep erythrocytes, 50 μg phytohemagglutinin and 25 μgpokeweed mitogen (Sigma) Immunostimulation was repeated 2 or 4 times atweekly intervals and cells were collected 3-4 days after lastimmunization. Blood was drained from tail-severed larvae, diluted 1:1with 0.57×PBS and 30 mM EDTA. Buffy coat leukocytes were collected after5 min centrifugation at 50 g. Cells were sorted using MoFlo cytometer asdescribed (Mayer et al., 2002a).

Subtracted Immunostimulated Lymphocyte cDNA Libraries

Super SMART PCR cDNA Synthesis (BD Biosciences) was used with mRNA fromlarge blood lymphocytes, myeloid cells and erythrocytes sorted fromlarvae immunostimulated 4 times at weekly intervals. Activatedlymphocyte cDNA was subtracted in 2 reactions against cDNA of myeloidcells or erythrocytes (PCR-Select, BD Biosciences). Subtracted productswere cloned in pGEM-T Easy (Promega) and 1,507 sequences were analyzed.

TABLE 3 PCR primers Primer Position Position (10 pmloe/μl)Sequence (5′-3′) (cDNA clone) (gVLR contig) Slit.F CTCGGCTCTGCAGCTCTCA 2-20 24872-24890 (SEQ ID NO: 159) (LRR-2913) LRR.F1 TGGCGCCCTGGTGCAAAGT153-171 25643-25661 (SEQ ID NO: 160) (LRR-2913) Slit.RGAACACTGCGAGGGACATG 179-197 25669-25687 (SEQ ID NO: 161) (LRR-2913)Dis_LRR.F AAAAGATCTTGTCCCTCGCAGTGTTC 181-197 (SEQ ID NO: 162) (LRR-2913)LRR.R1 ACGGACGGGGGTATTGGTA 633-651 37969-37987 (SEQ ID NO: 163)(LRR-2913) LRR_C.F1 ATCCCTGAGACCACCACCT 739-757 38075-38093(SEQ ID NO: 164) (LRR-2913) LRR_C.R1 CACGCCGATCAACGTTTCCT 928-94738264-38283 (SEQ ID NO: 165) (LRR-2913) Dis_LRR.R1AAAGTCGACACGCCGATCAACGTTTC 930-946 (SEQ ID NO: 166) (LRR-2913) LRR_C.R2CCGCCATCCCCGACCTTTG 948-966 38302-38284 (SEQ ID NO: 167) (LRR-2913)gVLR.F1 CCGGTTGGACACTAGTGTTG 22285-22304 (SEQ ID NO: 168) gVLR.R1GTGCCATTGGGATCAGTGGT 42099-42118 (SEQ ID NO: 169) GAPDH.FGAACATCGGCATCAATGGGT 71-90 (SEQ ID NO: 170) (PmGAPDH) GAPDH.RGAGGCCTTATCGATGGTGGT 366-385 (SEQ ID NO: 171) (PmGAPDH)

VLR RT-PCR

Buffy coat leukocytes from unstimulated larvae (#1-4), orimmunostimulated twice at one week intervals (#5-7), were pelleted 5 minat 300 g. First strand cDNA was primed with 50 ng random hexamers(SuperScript III; Invitrogen). VLR diversity regions were amplified withExpand High Fidelity (Roche) using LRR.F1+LRR.R1 (Table 3). Thermalcycling was as follows: 94° C. 1 min, then 35 cycles of 94° C. 30 sec,59° C. 30 sec, 72° C. 1 min. Per animal 10-12 clones were sequenced.

VLR Single Cell RT-PCR

Single lymphocytes, or a 10-cell pool, from buffy coats of unstimulatedlarva (#8), and one immunostimulated twice at one week interval (#9),were sorted into 0.2 ml TRIzol (Invitrogen). First strand cDNA wasprimed with LRR_C.R2. VLRs were amplified by 2 rounds of nested PCR,first Slit.F+LRR_C.R2 using Advantage II (BD Biosciences) thenLRRN_F1+LRR_C.R1 using Expand High Fidelity. Cycling parameters were:94° C. 1 min, then 40 cycles of 94° C. 30 sec, 60° C. 30 sec, 72° C. 1min. Colony PCR with vector primers revealed a single size insert in 6colonies from each of the 12 cells, 3 of which were sequenced. Coloniesfrom cell 9.16 revealed 2 sizes and 3 short and 3 long inserts weresequenced. From the pool of 10 unstimulated cells 6 clones weresequenced.

Genomic DNA and Genomic PCR

Genomic DNA was isolated from 1/3 whole larval body, erythrocytes from0.25 ml blood pelleted for 5 min at 50 g, or 107 sorted lymphocytes. PCRwas from 400 ng gDNA using Expand Long Template (Roche). VLR diversityregions were amplified from larvae #10 and 12, using LRR.F1+LRR.R1.Mature VLRs were amplified from animals #10 and 11, usingSlit.F+LRR_C.R2, or LRR_N.F1+LRR_C.R1. Amplification across the gVLR wasfrom animals #10 and 13, with gVLR.F1+gVLR.R1. The 8 kb band was clonedin pCR-XL (Invitrogen) and sequenced with: M13.Forward, M13.Reverse,Slit.F and LRR_C.R2.

Virtual Northern and DNA Blots

Virtual Northern was prepared as recommended (Super SMART manual).Twenty cycleamplified cDNA was from larval tail, liver and sortedlymphocytes from blood, typhlosole and kidneys of unstimulated animals,or small and large blood lymphocytes, myeloid cells and erythrocytessorted from blood of larvae immunostimulated 4 times at weeklyintervals.

Genomic DNA from larvae #10, 12 and 13, 10 μg per lane, was digestedwith BamHI, EcoRI or HindIII (Roche); 5 μg lymphocyte DNA was digestedwith EcoRI. For the pulse-field CHEF blot, erythrocytes from 10 larvaewere embedded in agarose, and 20 μg DNA per lane were digested withAscI, FseI, NotI, PacI, PmeI, or SfiI.

The following 32P-labled probes were used: VLR N-terminal probe, 196 bp,PCR amplified from clone LRR-2913 using Slit.F+Slit.R, and C-terminalprobe, 208 bp, amplified with LRR_C.F1+LRR_C.R1; GAPDH probe, 314 bp,amplified from clone PmGAPDH using GAPDH.F+GAPDH.R.

PAC Library and Clones

Arrayed sea lamprey PAC library in pCYPAC6 (AF133437) was constructedfrom erythrocyte DNA of one Lake Michigan adult using partial MboIdigests. The 6×104 clones had 65 kb average inserts with 1-2 fold genomecoverage. Library was screened using both N-terminal and Cterminalprobes. Plasmids of positive clones were EcoRI digested, blotted andhybridized either with the N-terminal or C-terminal probes. Five PACshybridized with both probes (2, 3, 15, 16, 17) and 5 PACs hybridizedonly with the N-terminal probe (4, 9, 14, 35, 42, 43).

The gVLR was amplified with Expand Long Template from plasmids of PACs2, 3, 15, 16 and 17 using Slit.F+LRR_C.R2. All PCR products were of 14kb, with 2 sets of BamHI patterns (PACs 2, 3 and 15-17). PACs 3, 4 and16 were sequenced at McGill University (Quebec, Canada).

VLR GPI-Anchor

A VLR insert, LRRNT to stop codon, was amplified from clone LRR-2913with Expand High Fidelity using Dis_LRR.F+Dis_LRR.R1 and fused to Igκsignal peptide and Hemagglutinin epitope in pDisplay (Invitrogen).Surface localization and VLR GPI-anchor were analyzed in BW1547 cells,or controls expressing mFcγRIIb. Cells were treated with 1 unit/mlbacterial GPlspecific phospholipase C (Sigma) 45 min at 30° C. Surfacestaining of epitope tagged proteins was with anti-HA-tag mAb 12CA5.

Sequence Analysis

Sequence variability was estimated using MEGA 2.1 UPGMA (Kumar et al.,2001). GPI-anchor site was identified via: http://129.194.185.165/dgpi/.SWISS-MODEL VLR 3D structure was via:http://cubic.bioc.columbia.edu/predictprotein/submit_meta.html. Residues22-319 fom clone 12.26 were threaded on crystal coordinates of CD42a (1m10.pdb) and NOGO-66 receptor (1p8t.pdb).

Example 2 Variable Lymphocyte Receptors in Hagfish Cyclostome VLRHomologs

Two distinct types of VLR, VLR-A and VLR-B, were identified amongexpressed sequence tags from 12,000 leukocyte cDNA clones of the Inshorehagfish, Eptatretus burgeri (Suzuki et al., 2004B). Matching VLR werethen cloned by RT-PCR from transcripts of lymphocyte-like cells of thePacific hagfish, E. stoutii. FIG. 9 depicts an alignment of the aminoacid sequences of hagfish VLR-A and VLR-B, the Sea lamprey VLR(Petromyzon marinus) and VLRs of two non-parasitic lampreys, Americanbrook lamprey (Lampetra appendix) and Northern brook lamprey(Ichthyomyzon fossor). These VLR share similar structural domains: asignal peptide (SP), N-terminal LRR (LRRNT), 18-residue LRR1 followed bya variable number of 24-residue LRRs, a 13-residue connecting peptide(CP) and C-terminal LRR (LRRCT). At the beginning of the C-terminus thelamprey VLR and hagfish VLR-B have a threonine/proline-rich region, butthis region is not well conserved in the hagfish VLR-A. All VLR proteinsend with a hydrophobic tail region that is required for modification ofthe protein to add a glycosyl-phosphatidyl-inositol (GPI) cell surfacemembrane anchor. Like the sea lamprey VLR, hagfish VLR-A was predictedto be a GPI-anchored protein although no co cleavage site was identified(DGPI http://129.194.185.165/dgpi/); the C-terminal hydrophobicityprofile for VLR-B is also predictive of GPI modification.

Transcripts of hagfish VLR are abundant in lymphocyte-like cells, butnot in myeloid cells or erythrocytes sorted by their light scattercharacteristics. VLR-A transcript levels were ˜3-fold higher than VLR-Blevels in blood leukocyte samples. Both VLR types of the Pacific hagfishare highly heterogeneous (FIGS. 10A and B), exhibiting variable numbersof the 24-residue LRR modules and pronounced LRR sequence diversity.Comparable diversity was observed for VLR-A (N=66) and VLR-B (N=18)sequences from Inshore hagfish (FIG. 13). Interestingly, five clustersof 2-4 VLR-A clones that were identical or differed by only 1-2 residueswere found among the 40 transcripts from hagfish #5 (marked by asterisksin FIG. 10A), that was given four weekly injections of an antigen andmitogen cocktail. The finding that 30% of the VLR-A transcripts fromthis hagfish consisted of clusters of related sequences indicates clonalexpansion of VLR-A bearing lymphocytes. The clones with 1-2 amino acidsubstitutions reflect additional VLR diversification through somatichypermutation.

The dataset of unique sequence Pacific hagfish VLR-A (N=130) reveals 2-6copies per transcript of the 24-residue LRRs (N=527; average 4). In theVLR-B dataset (N=69) there are 1-6 copies of the 24-residue LRRs (N=195;average 2.8), while in the set of 129 Sea lamprey VLR (19; GenBankaccessions AY577943-AY578059) there were 1-9 copies of 24-residue LRRs(N=325; average 2.5). The individual components of these VLR, except forLRRNT and LRRCT that were too diverse among the species for reliablealignment (Table 4; 328 LRR1 domains, 328 CP domains, and 1,047 singledomains of the 24-residue LRRs) were then analyzed separately in aNeighbor Joining phylogenetic tree.

TABLE 4 Components of unique hagfish and Sea lamprey VLR Unique LRRmotifs LRR1 (18 aa) CP (13 aa) Diversity LRR (24 aa) Diversity LRRconsensus* Es_VLR-A 77/130 (59%) 71/130 (55%) 477/527 (90%)-L--L--L-L--NqL--lP-G-FD (SEQ ID NO: 304) Es_VLR-B  68/69 (98%)  46/69(67%) 190/195 (97%) KLT-Lt-L-L--NqL-S-P-GvFD (SEQ ID NO: 305) Pm_VLR68/129 (53%) 36/129 (28%) 269/325 (83%) -L--L--L-L--NQL---P-G-FD (SEQ IDNO: 306) *Consensus—capital letters: 80-100% identity; small letters:60-79%The clusters were nearly exclusively of the same type and speciesorigin, i.e., Pacific hagfish VLR-A, VLR-B or Sea lamprey VLRclustering. There were no instances of identical LRR domains between thedifferent VLR types. However, a large portion of the LRR1 and CP domainswithin hagfish VLR-A and lamprey VLR clusters were identical (Table 4).In contrast, the LRR1 domains in hagfish VLR-B were 98% unique; the setsof 24-residue LRRs also consisted predominantly of unique sequences: 97%were unique in hagfish VLR-B, 90% in VLR-A and 83% in the Sea lampreyVLR. This remarkably high degree of diversity is especially remarkablegiven that consensus sequences derived for each of the 24-residue LRRtypes share at least 10 framework residues.

Hagfish VLR Genes

Genomic organization of the Pacific and Inshore hagfish VLR loci wasdetermined from sequences of large insert genomic clones isolated frombacterial artificial chromosome (BAC) libraries, one BAC for each VLRtype (FIG. 11). Only one copy of each of the gVLRs was identified inhagfish genomes. The sequences and organization of the loci are nearlyidentical in both species and fairly conserved between gVLR-A andgVLR-B. Hagfish gVLR begin with a 5′ untranslated region (UTR) that isfollowed by two coding regions (FIG. 12A). As in the Sea lamprey gVLR,the 5′ UTR is split by an intron, 6.4 kb long in the Pacific hagfishgVLR-A and 220 bp in gVLR-B. The first coding region in the hagfish gVLRencodes the signal peptide and an LRRNT domain in gVLR-A and onlyresidues 1-13 of the 23-residue signal peptide in gVLR-B. Next, thereare short intervening sequences of 171 and 211 bp for gVLR-A and gVLR-B,respectively. The second coding region consists of the 3′ end of LRRCTand the C-terminus, as in the Sea lamprey gVLR, except that the lampreyregion coding for the 5′ end of LRRCT is missing. The hagfish gVLR arecompact, 671 bp from start-to-stop codons in gVLR-A and 410 bp ingVLR-B.

The hagfish gVLR loci harbor cassettes encoding diverse LRR motifslocated ˜20-40 kb downstream from the germline genes (FIG. 11). In theVLR-A locus there is a cassette encoding 6-8 terminal residues of adiverse CP domain and a 5′ LRRCT that includes a 4-residue identicaloverlap with the gVLR-A 3′ LRRCT. Farther downstream there is a cassetteof two diverse LRRs positioned in reverse orientation relative to thegVLR-A and then an inverted incomplete 5′ LRRCT. In the gVLR-B locus,there is a cassette encoding residues 7-23 of the signal peptide and a5′ LRRNT, then a diverse CP domain and 5′ LRRCT, one inverted LRR and,farther downstream, another inverted LRR cassette consisting of the12-terminal residues and 8-proximal residues of LRRs. No other diverseLRR modules were identified in flanking DNA spanning ˜50 kb upstream and˜70 kb downstream from the gVLRs. However, diverse LRR elements likelyexist elsewhere in the genome to provide missing components of themature VLR genes identified in samples of genomic PCR amplicons fromlymphocyte-like cells: 35 unique mature VLR-A and 38 VLR-B sequencesfrom two animals (FIG. 10). Thus, the hagfish mature VLR genes must beassembled through somatic recombination, as is the case for lamprey.

Germline VLR genes in hagfish lymphocyte-like cells are activelytranscribed prior to gene rearrangement. PCR amplicons of VLR-A germlinetranscripts are ˜0.7 kb long and ˜0.5 kb for VLR-B (FIG. 12B, RT-PCR;position of PCR primers indicated in FIG. 12A) while the largeramplicons correspond to transcripts from the rearranged mature VLRgenes, ˜1.1 and ˜0.8 kb for VLR-A and VLR-B respectively. Thecorresponding PCR amplicons from blood genomic DNA are ˜0.7 kb for thegermline genes and ˜1.1 kb for the mature VLR-A and VLR-B genes (FIG.12B, genomic PCR). In transcripts from germline and mature VLR genes,the 5′ intron is spliced out to yield RT-PCR products shorter than thecorresponding genomic PCR amplicons (see VLR-B in FIG. 12B; gVLR-Aamplicons do not include the 6.4 kb intron). However, the interveningsequences between the coding exons are retained in the germlinetranscripts because they lack consensus eukaryotic splice sites. Thegermline transcription may be required for gVLR rearrangement, as is thecase in mammalian antibody class switch recombination for which germlineswitch region transcription is obligatory (Bottaro et al., 1994; Hein etal., 1998).

VLR Phylogeny

A phylogenetic analysis of the agnathan VLR proteins reveals threedistinct clusters respectively composed by lamprey VLR, hagfish VLR-Aand VLR-B sequences (FIG. 12C). The hagfish VLR-B and lamprey VLRcluster in a separate branch from that with the hagfish VLR-A. The sametree topology was seen when only the VLR diversity regions, LRRNT toLRRCT or LRR1 to CP, were aligned. Hence, either the hagfish VLR-A aroseby duplication of the ancestral gene (FIG. 12D) or the lamprey losttheir VLR-A ortholog after the split between the hagfish and lampreylineages, dating 499±38 Myr ago in the Cambrian period (Hedges et al.,2001). It is also possible that a lamprey VLR-A ortholog exists, but wasnot detected in >18,000 cDNA sequences derived from lampreylymphocyte-like cells (Pancer et al., 2004) because it is expressed atvery low levels or in non-lymphoid cells.

The presence of VLRs in both of the extant cyclostome orders isindicative of strong evolutionary pressure for vertebrates to develop ananticipatory molecular recognition system. The analysis indicates that,within less than 40 million years in the Cambrian, two radicallydifferent systems evolved in agnathans and gnathostomes in which eitherLRR or Ig gene fragments undergo recombinatorial assembly to generatediverse repertoires of lymphocyte receptors. This evolutionary scenarioraises many intriguing questions, one of which concerns the issue ofwhether the two adaptive immune strategies represent convergentevolution or if one was ancestral to the other. Whether VLRs wereforerunner vertebrate immune receptors or the rearranging VLRs and Igsevolved independently will become certain only with an unambiguousresolution of the phylogenetic relationships among the groups of livingand extinct jawless and jawed vertebrates (Mallatt et al., 2003; Meyeret al., 2003). In this regard, however, the presence of VLRs in bothorders of contemporary agnathans lends additional molecular evidencefavoring a monophyletic origin of cyclostomes.

Animals.

Live specimens of Pacific hagfish Eptatretus stoutii (30-60 cm long)were purchased form Marinus Scientific (Long Beach, Calif.) andmaintained for two months at 12° C. in artificial sea water (OceanicSystem, Dallas, Tex.). Larvae (15-20 cm long) of the American brooklamprey (Lampetra appendix) and Northern brook lamprey (Ichthyomyzonfossor), were from tributaries to the Great Lakes (Lamprey Services,Ludington, Mich.).

Hagfish were sedated by immersion for 15 min in 0.5 gr/liter MS222(Sigma, St. Louis, Mo.) buffered to pH=7 before intraperitonealinjection with an antigen/mitogen cocktail in 0.5 ml hagfish PBS (perlitter: 28 gr NaCl, 0.2 gr KCL, 1.44 gr Na₂HPO₄, 0.24 gr KH₂PO₄, pH=7.4,1 osmole). The cocktail contained 10⁹ live E. coli TG1 bacteria, 10⁹sheep erythrocytes (Colorado Serum Company, Denver, Colo.) and 100 μgeach phytohemagglutinin and pokeweed mitogen (Sigma) Immune stimulationwas repeated at weekly intervals and four days after the fourthstimulation blood was collected with a syringe from the tail blood sinusand diluted 1:1 with hagfish PBS containing 30 mM EDTA. Buffy coatleukocytes collected after 5 min centrifugation at 50×g were sorted bytheir light scatter characteristics as described (Newton et al., 1994;Raison et al., 1994) using a MoFlo cytometer (Cytomation, Fort Collins,Colo.).

Hagfish VLR.

Inshore hagfish Eptatretus burgeri VLR homologs were identified usinglamprey VLR as BLAST queries against the database of expressed sequencetags from leukocyte RNA of unstimulated animals #7,8 (Suzuki et al.,2004B). Clones with significant matches were sequenced on both strands:64 VLR-A and 15 VLR-B cDNA clones. For the Pacific hagfish, unseparatedblood cells and buffy coat leukocytes from three unstimulatedindividuals (#1-3,6), and buffy coat leukocytes from twoimmunostimulated animals (#4,5) were used for extraction of bloodgenomic DNA and leukocyte RNA. Extraction of RNA was with TRIzol Reagent(Invitrogen, Carlsbad, Calif.) and PolyA RNA was selected with DynabeadsmRNA purification Kit (Dynal Biotech, Lake Success, N.Y.). First strandcDNA synthesis was primed with 20 pmoles of the HgVLRA.F1 (Table 5) forVLR-A, or HgVLRB.F1 for VLR-B, using SuperScript III First Strand cDNASynthesis kit (Invitrogen), and the products were column purified(QIAquick PCR purification; QIAGEN, Valencia, Calif.).

TABLE 5 VLR PCR primers Position in  Position in Name Sequence 5′-3′cDNA clone Eb_gVLR Contig HgVLRA.F1 TGGTGATAACCTCAAGGTGCT 35-559597-9614 (SEQ ID NO: 322) (Eb7VLRA.21) HgVLRA.F2 CAGAGATGATGGGTCCGGT60-78 15509-15527 SEQ ID NO: 323) (Eb7VLRA.21) HgVLRA.R1GGCAAGTGAGACACTGGTTC 1023-1042 16166-16185 (SEQ ID NO: 324) (Eb7VLRA.21)HgVLRA.R2 TCTTGAGAAAGTGGAAGACGTA  995-1016 16138-16159 (SEQ ID NO: 325) Eb7VLRA.21) HgVLRB.F1 CACGAGGATTGCACGTGAAGA 49-69 59421-59441(SEQ ID NO: 326) (Eb7VLRB.15) HgVLRB.F2 TTCCACCTCGAGGAAGATGA  93-11259677-59696 (SEQ ID NO: 327) (Eb7VLRB.15) HgVLRB.R1 GGCAAAATGTTGGACGGTGT866-885 60116-60135 (SEQ ID NO: 328) (Eb7VLRB.15) HgVLRB.R2GGCGTGACATATGAGGTAAAC 826-846 60076-60096 (SEQ ID NO: 329) (Eb7VLRB.15)Slit.F CTCGGCTCTGCAGCTCTCA   1-19 (SEQ ID NO: 330) (LaVLR.2) LRR_N.F1CTCCGCTACTCGGCCTGCA   1-19 (SEQ ID NO: 331) (IfVLR.15) VLR_3UT.RGATGAAGCGAAGACAGACGTG 1607-1627 (SEQ ID NO: 332) (LaVLR.2) VLR_3UT.RGATGAAGCGAAGACAGACGTG 1405-1425 (SEQ ID NO: 333) (IfVLR.15)VLRs were then PCR amplified using Expand High Fidelity PCR (RocheApplied Science, Indianapolis, Ind.), from the cDNA or from genomic DNA,in 50 μl reactions containing: 1 μl each of the sets of forward andreverse primers (F1 or F2 and R1 or R2) at 10 pmole/μl, 5 μl 10× buffer,36.25 μl DDW, 5 μl cDNA or genomic DNA (250 ng) and 0.75 μl Expandenzyme. Reactions were amplified using one cycle of 94° C. 1 min, then35 cycles of 94° C. 30 sec, 58° C. 30 sec and 72° C. 1 min, and a final7 min elongation at 72° C. Products were column purified, cloned inpCRII-TOPO (Invitrogen) and the inserts were sequenced. For the Pacifichagfish, 109 VLR-A RT-PCR clones were sequenced (four contained in-framestop codons), and 36 genomic mature VLR-A amplicons (two containedin-frame stop codons). For VLR-B, 37 RT-PCR clones were sequenced (onecontained an in-frame stop codon), and 38 genomic mature VLR-B amplicons(four contained in-frame stop codons). Liver genomic DNA from Inshorehagfish #9 (Suzuki et al., 2004B) was used for PCR cloning andsequencing mature VLRs: 4 mature VLR-A amplicons (two contained in-framestop codons) and 3 mature VLR-B amplicons.

Non-Parasitic Lamprey VLR

First strand cDNA was synthesized as above using the reverse primerVLR_(—)3UT.R (Sea lamprey 3′ UTR primer, Table 5). For the Americanbrook lamprey the forward primer was Slit.F (Sea lamprey 5′ UTR primer),and for the Northern brook lamprey LRR_N.F1 (another Sea lamprey 5′ UTRprimer). In total 13 unique VLR clones of the American brook lamprey andseven of the Northern brook lamprey were sequenced.

BAC Libraries and Clones.

An Inshore hagfish BAC library (Suzuki et al., 2004A) was screened byPCR using VLR primers as above (F1 or F2 and R1 or R2). The Pacifichagfish BAC library (VMRC23) was constructed from EcoRI partial digestsof erythrocyte DNA from a single specimen in the vector pCCBACE1(Epicentre Technologies, Madison Wis.). This library consists of˜184,000 recombinants and encompasses ˜5× coverage of the hagfishgenome. The entire library was screened by hybridization with 5′ and 3′VLR-A and VLR-B probes and positive clones were authenticated by PCR.One BAC for each VLR type from the Pacific and Inshore hagfish weresequenced at ˜10× coverage and assembled into contigs (Macrogen, Seoul,Korea). In case of incomplete sequence of the inserts only portionscontaining the gVLR and LRR cassettes were included with uncaptured gapsin the contigs: Eb_gVLR-A, 43,362 bp; Eb_gVLR-B, 92,072 bp; Es_gVLR-A,81,648 bp; Es_gVLR-B, 76,730 bp.

Sequence Analysis

Neighbor Joining and UPGMA trees were constructed with the pairwisedeletion option using the programs from MEGA 3 Molecular EvolutionaryGenetics Analysis (Kumar et al., 2004). Prediction of genes in the BACinserts was accomplished by using local BLAST downloaded fromftp://ftp.ncbi.nlm.nih.gov/blast/executables/ and the GenScan server:genes.mit.edu/GENSCAN.html.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Thereferences disclosed are also individually and specifically incorporatedby reference herein for the material contained in them that is discussedin the sentence in which the reference is relied upon.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

REFERENCES

-   Anderson M K, Sun X, Miracle A L, Litman G W and Rothenberg E    V (2001) Evolution of hematopoiesis: Three members of the PU.1    transcription factor family in a cartilaginous fish, Raja    eglanteria. Proc. Natl. Acad. Sci. USA 98:553-8-   Ardavin CF and Zapata A (1987) Ultrastructure and changes during    metamorphosis of the lympho-hemopoietic tissue of the larval    anadromous sea lamprey Petromyzon marinus. Dev. Comp. Immunol.,    11:79-93-   Azumi K et al.,. Genomic analysis of immunity in a Urochordate and    the emergence of the vertebrate immune system: “waiting for Godot”.    Immunogenetics 55: 570-81, 2003-   Bell, J K., Mullen, G E D., Leifer, C A. Mazzoni, A., Davies, D R.    and Segal, D M. Leucine-rich repeats and pathogen recognition in    Toll-like receptors. Trends in Immunology 2003, 24: 528-533.-   Beutler, B. Innate immunity: an overview. Molecular Immunology    40 (2004) 845-859. Bryan, M. B., Libants, S. V., Warrillow, J. A.,    Li, W. and Scribner, K. T. Polymorphic microsatellite markers for    the landlocked sea lamprey, Petromyzon marinus. Conservation    Genetics 4: 113-116, 2003-   Bottaro, A., Lansford, R., Xu, L., Zhang, J., Rothman, P. &    Alt, F. W. (1994) EMBO J. 13, 665-674.-   Chamaillard, M., Girardin, S E., Viala, J. and Philpott, D J. Nods,    Nalps and Naip: intracellular regulators of bacterial-induced    inflammation. Cellular Microbiology (2003) 5: 581-592.-   Cooper A J (1971) Ammocoete lymphoid cell populations in vitro. In:    4th Leukocyte Culture Conference. O. R. McIntyre (Ed). New York    Appleton Century-Crofts. pp. 137-47-   Donelson J E. Antigenic variation and the African trypanosome    genome. Acta Trop. 2003, 85: 391-404.-   Finstad J and Good R A (1964) The evolution of the immune    response. III. Immunologic responses in the lamprey. J. Exp. Med.,    120: 1151-67-   Finstad J, Papermaster B W and Good R A (1964) Evolution of the    immune response. II. Morphologic studies of the thymus and organized    lymphoid tissue. Lab Invest., 13:490-512-   Flajnik M F and Kasahara M (2001) Comparative genomics of the MHC:    glimpses into the evolution of the adaptive immune system. Immunity    15:351-62-   Flajnik M F (2002) Comparative analyses of immunoglobulin genes:    surprises and portents. Nat. Rev. Immunol., 2:688-98-   Forey P L and Janvier P (1993) Agnathans and the origin of jawed    vertebrates. Nature 361:129-134-   Fujii T (1982) Electron microscopy of the leukocytes of the    typhlosole in ammocoetes, with special attention to the    antibody-producing cells. J. Morphol., 173:87-100-   Fujii T and Hayakawa I (1983) A histological and    electron-microscopic study of the cell types involved in rejection    of skin allografts in ammocoetes. Cell Tissue Res., 231:301-12-   Good, R. A., Finstad, J. & Litman, G. W. in The biology of lampreys    II: Immunology (Eds Hardisty, M. V. & Potter, I. C.) 405-432    (Academic Press, London 1972).-   Grimholt U, Larsen S, Nordmo R, Midtlyng P, Kjoeglum S, Storset A,    Saebo S, Stet R J. MHC polymorphism and disease resistance in    Atlantic salmon (Salmo salar); facing pathogens with single    expressed major histocompatibility class I and class II loci.    Immunogenetics. 55:210-9, 2003-   Hagen M, Filosa M F and Youson J H (1985) The immune response in    adult sea lamprey (Petromyzon marinus L.): the effect of    temperature. Comp. Biochem. Physiol., 82:207-10-   Haire R N, Miracle A L, Rast J P and Litman G W (2000) Members of    the Ikaros gene family are present in early representative    vertebrates. J. Immunol., 165:306-12-   Hamrick T S, Dempsey J A, Cohen M S, Cannon J G. Antigenic variation    of gonococcal pilin expression in vivo: analysis of the strain    FA1090 pilin repertoire and identification of the pilS gene copies    recombining with pilE during experimental human infection.    Microbiology 2001, 147: 839-49.-   Hedges, S. B. (2001) in Major events in early vertebrate evolution,    Systematics Association special vol. 61: Molecular evidence for the    early history of living vertebrates, ed Ahlberg, P. E.(Taylor &    Francis, London), pp. 119-134.-   Hein, K., Lorenz, M. G., Siebenkotten, G., Petry, K., Christine, R.    & Radbruch, A (1998) J. Exp. Med. 188, 2369-2374.-   Ikezawa, H. Glycosylphosphatidylinositol (GPI)-Anchored Proteins.    Biol. Pharm. Bull. 25: 409-417 (2002)-   Jones, D. A. and Takemoto, D. Plant innate immunity—direct and    indirect recognition of general and specific pathogen-associated    molecules. Current Opinion in Immunology 2004, 16:48-62-   Kaufman J (2002) The origins of the adaptive immune system: whatever    next? Nat. Immunol., 3:1124-5-   Kilarski W and Plytycz B (1981) The presence of plasma cells in the    lamprey (Agnatha). Dev. Comp. Immunol., 5:361-6-   Kumar, S., Tamura, K., Jakobsen, I. B. and Nei, M. (2001) MEGA2:    Molecular Evolutionary Genetics Analysis software, Arizona State    University, Tempe, A R Laird D J, De Tomaso A W, Cooper M D and    Weissman I L (2000) 50 million years of chordate evolution: seeking    the origins of adaptive immunity. Proc. Natl. Acad. Sci., USA    97:6924-6-   Kumar, S., Tamura, K. & Nei, M. (2004) Brief Bioinform. 5, 150-163.-   Landmann, R., Müller, B. and Zimmerli, W. CD14, new aspects of    ligand and signal diversity. Microbes and Infection, 2, 2000,    295-304.-   Litman G W, Frommel D, Finstad F J, Howell J, Pollara B W and Good R    A (1970) The evolution of the immune response. VIII. Structural    studies of the lamprey immunoglobulin. J. Immunol., 105:1278-85-   Mallatt, J. & Chen, J. Y. (2003) J. Morphol. 258, 1-31.-   Marchalonis J J and Edelman G M (1968) Phylogenetic origins of    antibody structure. 3. Antibodies in the primary immune response of    the sea lamprey, Petromyzon marinus. J. Exp. Med., 127:891-914-   Mayer W E, Uinuk-Ool T, Tichy H, Gartland L A, Klein J and Cooper M    D (2002 a) Isolation and characterization of lymphocyte-like cells    from a lamprey. Proc. Natl. Acad. Sci., USA 99:14350-5-   Mayer W E, O'Huigin C, Tichy H, Terzic J and Saraga-Babic M (2002 b)    Identification of two Ikaros-like transcription factors in lamprey.    Scand. J. Immunol., 55:162-70-   Meyer, A. & Zardoya, R. (2003) Annu. Rev. Ecol. Evol. Syst. 34,    311-338.-   Newton, R. A., Raftos, D. A., Raison, R. L. & Geczy, C. L. (1994)    Dev. Comp. Immunol. 18, 295-303.-   Pancer, Z., Mayer, W. E., Klein, J. & Cooper, M. D. (2004) Proc.    Natl. Acad. Sci. USA 101, 13273-13278.-   Penn D J, Damjanovich K, Potts W K. MHC heterozygosity confers a    selective advantage against multiple-strain infections. Proc Natl    Acad Sci 99:11260-42002, 2002-   Perey D Y, Finstad J, Pollara B and Good R A (1968) Evolution of the    immune response. VI. First and second set skin homograft rejections    in primitive fishes. Lab. Invest., 19:591-7-   Piavis G W and Hiatt J L (1971) Blood cell lineage in the sea    lamprey Petromyzon marinus (Pisces: Petromyzontidae). Copeia 4:722-8-   Pollara B, Litman G W, Finstad J, Howell J and Good R A (1970) The    evolution of the immune response. VII. Antibody to human “O” cells    and properties of the immunoglobulin in lamprey. J. Immunol.,    105:738-45-   Raison, R. L., Coverley, J., Hook, J. W., Towns, P., Weston, K. M. &    Raftos, D. A (1994) Immunol. Cell Biol. 72, 326-332.-   Rast, J. P., Michele K. Anderson, M. K., Strong, S. J., Luer, C.,    Litman, R. T., and Litman, G. W. α, β, g, and δ T Cell Antigen    Receptor Genes Arose Early in Vertebrate Phylogeny. Immunity,    6:1-11, 1997.-   Schluter S F, Bernstein R M, Bernstein H and Marchalonis J J (1999)    ‘Big Bang’ emergence of the combinatorial immune system. Dev. Comp.    Immunol., 23:107-11-   Schwede, T., Diemand, A. Guex, N. and Peitsch, M. V. Protein    structure computing in the genomic era. Research in Microbiology    151: 107-112 (2000)-   Shintani S, Terzic J, Sato A, Saraga-Babic M, O'hUigin C, Tichy H    and Klein J (2000) Do lampreys have lymphocytes? The Spi evidence.    Proc. Natl. Acad. Sci., USA 97:7417-22-   Suzuki, T., Ota, T., Fujiyama, A. & Kasahara, M. (2004A) Genes    Genet. Syst. 79, 251-253.-   Suzuki, T., Shin-I, T., Kohara, Y. & Kasahara, M. (2004B) Dev. Comp.    Immunol. 28, 993-1003.-   Uinuk-Ool T, Mayer W E, Sato A, Dongak R, Cooper M D and Klein    J (2002) Lamprey lymphocyte-like cells express homologs of genes    involved in immunologically relevant activities of mammalian    lymphocytes. Proc. Natl. Acad. Sci., USA 99:14356-61-   Uinuk-Ool T S, Mayer W E, Sato A, Takezaki N, Benyon L, Cooper M D    and Klein J (2003) Identification and characterization of a    TAP-family gene in the lamprey. Immunogenetics 55:38-48-   Wang D, Botkin D J, Norris S J. Characterization of the vls    antigenic variation loci of the Lyme disease spirochaetes Borrelia    garinii Ip90 and Borrelia afzelii ACAI. Mol. Microbiol. 2003, 47:    1407-17.-   Zapata A, Ardavin C F, Gomariz R P and Leceta J (1981) Plasma cells    in the ammocoete of Petromyzon marinus. Cell Tissue Res., 221:203-8.

1-23. (canceled)
 24. An isolated nucleic acid that encodes a polypeptidecomprising an N-terminal leucine rich repeat (LRRNT), one or moreleucine rich repeats (LRRs), a C-terminal leucine rich repeat (LRRCT),and a connecting peptide, wherein the connecting peptide comprises analpha helix and wherein the isolated polypeptide is a variablelymphocyte receptor (VLR), and wherein the VLR selectively binds anantigen and wherein the VLR can function in an adaptive immunity and canbe generated by somatic rearrangement.
 25. An expression vectorcomprising the nucleic acid of claim 24 operably linked to an expressioncontrol sequence.
 26. A cultured cell comprising the vector of claim 25.27-50. (canceled)
 51. The nucleic acid of claim 24, wherein theconnecting peptide is linked to the LRRCT.
 52. The nucleic acid of claim24, wherein the polypeptide further comprises a stalk region and aglycosyl-phosphatidyl-inositol anchor.
 53. The nucleic acid of claim 52,wherein the polypeptide further comprises a hydrophobic tail.
 54. Thenucleic acid of claim 52, wherein the stalk region comprises athreonine-proline rich region.
 55. The nucleic acid of claim 24, whereinthe polypeptide further comprises a signal peptide.
 56. The nucleic acidof claim 24, wherein the polypeptide comprises 1-9 LRRs, with LRR1adjacent to LRRNT.
 57. The nucleic acid of claim 56, wherein LRR1comprises less than about 20 amino acids.
 58. The nucleic acid of claim56, wherein LRR1 comprises about 18 amino acids.
 59. The nucleic acid ofclaim 56, wherein each of LRR2-9 comprises less than about 25 aminoacids.
 60. The nucleic acid of claim 24, wherein the LRRNT comprisesless than about 40 amino acids.
 61. The nucleic acid of claim 60,wherein the LRRNT comprises the amino acid sequence of SEQ ID NO:157.62. The nucleic acid of claim 60, wherein the LRRNT comprises the aminoacid sequence of SEQ ID NO:157 with one or more conservative amino acidsubstitutions.
 63. The nucleic acid of claim 24, wherein the LRRCTcomprises less than about 60 amino acids.
 64. The nucleic acid of claim63, wherein the LRRCT comprises the amino acid sequence of SEQ IDNO:158.
 65. The nucleic acid of claim 63, wherein the LRRCT comprisesthe amino acid sequence of SEQ ID NO:158 with one or more conservativeamino acid substitutions.
 66. The nucleic acid of claim 24, wherein theconnecting peptide comprises less than about 15 amino acids.
 67. Thenucleic acid of claim 24, wherein the LRRs differ in amino acid sequencefrom each other and from the LRRNT and the LRRCT.
 68. The nucleic acidof claim 24, wherein the polypeptide is about 130 to about 225 aminoacids in length.
 69. The nucleic acid of claim 24, wherein the antigenis a pathogen.
 70. The nucleic acid of claim 69, wherein the pathogen isa bacterium.
 71. The nucleic acid of claim 24, wherein the antigen is atoxin.